Shizhong Zheng

Curcumin attenuates angiogenesis in liver fibrosis and inhibits angiogenic properties of hepatic stellate cells

Hepatic fibrosis is concomitant with sinusoidal pathological angiogenesis, which has been highlighted as novel therapeutic targets for the treatment of chronic liver disease. Our prior studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work demonstrated that curcumin ameliorated fibrotic injury and sinusoidal angiogenesis in rat liver with fibrosis caused by carbon tetrachloride. Curcumin reduced the expression of a number of angiogenic markers in fibrotic liver. Experiments in vitro showed that the viability and vascularization of rat liver sinusoidal endothelial cells and rat aortic ring angiogenesis were not impaired by curcumin. These results indicated that hepatic stellate cells (HSCs) that are characterized as liver‐specific pericytes could be potential target cells for curcumin. Further investigations showed that curcumin inhibited VEGF expression in HSCs associated with disrupting platelet‐derived growth factor‐β receptor (PDGF‐βR)/ERK and mTOR pathways. HSC motility and vascularization were also suppressed by curcumin associated with blocking PDGF‐βR/focal adhesion kinase/RhoA cascade. Gain‐ or loss‐of‐function analyses revealed that activation of peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) was required for curcumin to inhibit angiogenic properties of HSCs. We concluded that curcumin attenuated sinusoidal angiogenesis in liver fibrosis possibly by targeting HSCs via a PPAR‐γ activation‐dependent mechanism. PPAR‐γ could be a target molecule for reducing pathological angiogenesis during liver fibrosis.

Cryptotanshinone Activates p38/JNK and Inhibits Erk1/2 Leading to Caspase-Independent Cell Death in Tumor Cells

Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism is not well understood. Here, we show that CPT induced caspase-independent cell death in human tumor cells (Rh30, DU145, and MCF-7). Besides downregulating antiapoptotic protein expression of survivin and Mcl-1, CPT increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK), and inhibited phosphorylation of extracellular signal–regulated kinases 1/2 (Erk1/2). Inhibition of p38 with SB202190 or JNK with SP600125 attenuated CPT-induced cell death. Similarly, silencing p38 or c-Jun also in part prevented CPT-induced cell death. In contrast, expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) conferred resistance to CPT inhibition of Erk1/2 phosphorylation and induction of cell death. Furthermore, we found that all of these were attributed to CPT induction of reactive oxygen species (ROS). This is evidenced by the findings that CPT induced ROS in a concentration- and time-dependent manner; CPT induction of ROS was inhibited by N-acetyl-l-cysteine (NAC), a ROS scavenger; and NAC attenuated CPT activation of p38/JNK, inhibition of Erk1/2, and induction of cell death. The results suggested that CPT induction of ROS activates p38/JNK and inhibits Erk1/2, leading to caspase-independent cell death in tumor cells. Cancer Prev Res; 5(5); 778–87. ©2012 AACR.

Tetramethylpyrazine induces G0/G1 cell cycle arrest and stimulates mitochondrial-mediated and caspase-dependent apoptosis through modulating ERK/p53 signaling in hepatic stellate cells in vitro

Activation of hepatic stellate cells (HSCs) is a pivotal event in the pathogenesis of liver fibrosis. Pharmacological induction of HSC apoptosis could be a promising strategy for fibrosis regression. Natural product tetramethylpyrazine (TMP) exhibits potent antifibrotic activities in vivo. However, the molecular mechanisms remain to be defined. The present study aimed at investigating the anti-proliferative and pro-apoptotic effects of TMP on HSCs and elucidating the underlying mechanisms. Our results demonstrated that TMP had no apparent cytotoxic effects on hepatocytes, but significantly inhibited HSC proliferation and induced cell cycle arrest at the G0/G1 checkpoint. These effects were associated with TMP regulation of cyclin D1, p21, p27 and p53. Furthermore, we found that TMP disrupted mitochondrial functions and led to activation of caspase cascades in HSCs. Mechanistic investigations revealed that TMP selectively blocked the extracellular signal-regulated kinase (ERK) signaling and activated p53, which was required for TMP induction of caspase-dependent mitochondrial apoptosis in HSCs. Autodock simulations predicted that TMP could directly bind to ERK2 with two hydrogen bonds and low energy score, indicating that ERK2 could be a direct target molecule for TMP within HSCs. Moreover, TMP altered expression of some marker proteins relevant to HSC activation. These data collectively revealed that TMP modulation of ERK/p53 signaling led to mitochondrial-mediated and caspase-dependent apoptosis in HSCs in vitro. These studies provided mechanistic insights into the antifibrotic properties of TMP that may be exploited as a potential option for hepatic fibrosis.

Dll4-Notch signaling in regulation of tumor angiogenesis

Abstract Tumor angiogenesis is a complex process and involves the tight interplay of tumor cells, endothelial cells, phagocytes and their secreted factors, which may act as promoters or inhibitors of angiogenesis. Many signaling pathways involved in these processes such as vascular endothelial growth factor (VEGF), fibroblast growth factors, Wnt and mTOR signaling pathway. Though research has confirmed that VEGF can play an important role in tumor angiogenesis, and has designed a lot of drugs that target VEGF, both experimental and clinical studies showed that these pathways mentioned above including VEGF did not play key roles in tumor angiogenesis. With the deepening of the research, people find that of all the signaling pathways involved in tumor angiogenesis, Notch signaling is the most notable one and plays crucial role in tumor angiogenesis. It was previously recognized that the Notch signaling plays a key role only in physiological angiogenesis such as development, wound healing and pregnancy. However, an increasing number of studies have proved that Notch signaling is also involved in pathological angiogenesis such as tumor angiogenesis and plays a critical role in these processes. More importantly, compared to resistance caused by anti-VEGF or other signaling pathways, experimental evidence revealed that Notch was involved in anticancer drug resistance, indicating that targeting Notch could be a novel therapeutic approach to the treatment for cancer by overcoming drug resistance of cancer cells. More recently, research has demonstrated that Notch ligands Delta-like 4 (Dll4) plays a key role in tumor angiogenesis. Data show that Dll4 functions as a negative regulator of tumor angiogenesis and is upregulated in tumor vasculature. This review focus on recent insights into Dll4-Notch signaling in tumor angiogenesis and its mechanisms, which may be utilized for a potential pharmacological use as a target for anti-angiogenic cancer therapy.

Peroxisome proliferator-activated receptor-γ as a therapeutic target for hepatic fibrosis: from bench to bedside.

Hepatic fibrosis is a dynamic chronic liver disease occurring as a consequence of wound-healing responses to various hepatic injuries. This disorder is one of primary predictors for liver-associated morbidity and mortality worldwide. To date, no pharmacological agent has been approved for hepatic fibrosis or could be recommended for routine use in clinical context. Cellular and molecular understanding of hepatic fibrosis has revealed that peroxisome proliferator-activated receptor-γ (PPARγ), the functioning receptor for antidiabetic thiazolidinediones, plays a pivotal role in the pathobiology of hepatic stellate cells (HSCs), whose activation is the central event in the pathogenesis of hepatic fibrosis. Activation of PPARγ inhibits HSC collagen production and modulates HSC adipogenic phenotype at transcriptional and epigenetic levels. These molecular insights indicate PPARγ as a promising drug target for antifibrotic chemotherapy. Intensive animal studies have demonstrated that stimulation of PPARγ regulatory system through gene therapy approaches and PPARγ ligands has therapeutic promise for hepatic fibrosis induced by a variety of etiologies. At the same time, thiazolidinedione agents have been investigated for their clinical benefits primarily in patients with nonalcoholic steatohepatitis, a common metabolic liver disorder with high potential to progress to fibrosis and liver-related death. Although some studies have shown initial promise, none has established long-term efficacy in well-controlled randomized clinical trials. This comprehensive review covers the 10-year discoveries of the molecular basis for PPARγ regulation of HSC pathophysiology and then focuses on the animal investigations and clinical trials of various therapeutic modalities targeting PPARγ for hepatic fibrosis.

Curcumin modulates cannabinoid receptors in liver fibrosis in vivo and inhibits extracellular matrix expression in hepatic stellate cells by suppressing cannabinoid receptor type-1 in vitro.

Activation of hepatic stellate cells (HSCs) is a pivotal event leading to extracellular matrix (ECM) overproduction during hepatic fibrogenesis. Compelling evidence indicates that cannabinoid receptors (CBRs) play an important role in chronic liver disease. Antagonism of hepatic CBR type 1 (CBR1) could be a novel therapeutic strategy for liver fibrosis. Our previous studies have demonstrated that curcumin has potent antifibrotic activity, but the mechanisms remain to be elucidated. The current work was to examine the curcumin effect on CBRs system and its relevance to inhibition of ECM expression in HSCs. Our in vivo data demonstrated that curcumin ameliorated fibrotic injury, and downregulated CBR1 but upregulated CBR2 at both mRNA and protein levels in rat fibrotic liver caused by carbon tetrachloride. The subsequent in vitro investigations showed that curcumin reduced the mRNA and protein abundance of CBR1 in cultured HSCs and decreased the expression of three critical ECM proteins. Further analyses revealed that CBR1 agonist abrogated the curcumin inhibition of ECM expression, but CBR1 antagonist mimicked and reinforced the curcumin effects. Autodock simulations predicted that curcumin could bind to CBR1 with two hydrogen bonds. Collectively, our current studies revealed that curcumin reduction of liver fibrosis was associated with modulation of CBRs system and that antagonism of CBR1 contributed to curcumin inhibition of ECM expression in HSCs.

Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells by interrupting platelet-derived growth factor-β receptor-mediated ERK and p38 pathways.

Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with accumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotal event during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fibrosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSC activation stimulated by hydrogen peroxide (H(2)O(2)), an in vitro model mimicking the oxidative stress in liver fibrogenesis, and to elucidate the possible mechanisms. Our results demonstrated that H(2)O(2) at 5μM significantly stimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazine dose-dependently suppressed proliferation and induced apoptosis in H(2)O(2)-activated HSCs, and attenuated expression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regulated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidence suggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βR and might be involved in ligustrazine reduction of fibrotic marker gene expression under H(2)O(2) stimulation. Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H(2)O(2)-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by oxidative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis.

Danshensu has anti-tumor activity in B16F10 melanoma by inhibiting angiogenesis and tumor cell invasion.

Danshensu, the major water-soluble component of Radix Salviae Miltiorrhizae (Danshen), is the basic chemical structure of various salvianolic acids. This study was to evaluate the anti-tumor activity of danshensu in a series of in vitro and in vivo models. The effect of danshensu on B16F10 melanoma cell and HUVEC proliferation were assessed by MTS assay, and cell invasion and migration were investigated by transwell chamber assay. The effect of danshensu on angiogenesis was evaluated by HUVEC migration assay, tube formation assay and chick chorioallantoic membrane assay. The expression of MMP-2, -9 and VEGF in B16F10 melanoma cell were detected by western blotting after danshensu treatment. The role of danshensu in tumor metastasis in vivo was evaluated by spontaneous and experimental B16F10 melanoma metastasis model. Although danshensu had no inhibitory effect on B16F10 melanoma cell and HUVEC proliferation, it significantly inhibited B16F10 melanoma cell invasion (at 0.05, 0.5, 5 microM) and migration (at 0.5, 5 microM). It also dramatically suppressed VEGF-induced endothelial migration (at 0.5, 5 microM), tube formation in vitro (at 4, 20 microM) and new vessel formation in CAM in vivo (100 microg/egg). Danshensu (at 5, 50 microM) significantly down-regulates protein expression of MMP-2, -9 and VEGF in B16F10 melanoma cell. In animal model, danshensu (20, 40 mg/kg) also possessed inhibitory effect on lung metastasis in spontaneous (46-day treatment) and experimental (23-day treatment) B16F10 melanoma metastasis model. All these results suggest that danshensu has anti-tumor activity by affecting on tumor angiogenesis and tumor invasion.

Xanthatin Induces Cell Cycle Arrest at G2/M Checkpoint and Apoptosis via Disrupting NF-κB Pathway in A549 Non-Small-Cell Lung Cancer Cells

Xanthatin, a natural sesquiterpene lactone, has significant antitumor activity against a variety of cancer cells, yet little is known about its anticancer mechanism. In this study, we demonstrated that xanthatin had obvious dose-/time-dependent cytotoxicity against the human non-small-cell lung cancer (NSCLC) cell line A549. Flow cytometry analysis showed xanthatin induced cell cycle arrest at G2/M phase. Xanthatin also had pro-apoptotic effects on A549 cells as evidenced by Hoechst 33258 staining and annexin V-FITC staining. Mechanistic data revealed that xanthatin downregulated Chk1, Chk2, and phosphorylation of CDC2, which contributed to the cell cycle arrest. Xathatin also increased total p53 protein levels, decreased Bcl-2/Bax ratio and expression of the downstream factors procaspase-9 and procaspase-3, which triggered the intrinsic apoptosis pathway. Furthermore, xanthatin blocked phosphorylation of NF-κB (p65) and IκBα, which might also contribute to its pro-apoptotic effects on A549 cells. Xanthatin also inhibited TNFα induced NF-κB (p65) translocation. We conclude that xanthatin displays significant antitumor effects through cell cycle arrest and apoptosis induction in A549 cells. These effects were associated with intrinsic apoptosis pathway and disrupted NF-κB signaling. These results suggested that xanthatin may have therapeutic potential against NSCLC.

Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis induced by Vascular Endothelial Growth Factor.

Angiogenesis is crucial for tumor metastasis, with many compounds that inhibit tumor metastasis acting through suppression of angiogenesis. We investigated anti-angiogenic properties of Ligustrazine in a series of in vitro and in vivo models. Ligustrazine inhibited VEGF-induced HUVECs migration and tube formation in a dose-dependent manner in vitro, and had limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 microg/ml. Ligustrazine also suppressed VEGF-induced rat aortic ring sprouting dose-dependently. Invivo, Ligustrazine reduced the Hb content in a Matrigel plug implanted in mice and inhibited new vessel formation in CAM. In addition, in a B16F10 spontaneous metastasis model, Ligustrazine decreased the expression of CD34 and VEGF in primary tumor tissue and reduced the number of metastasis nodi on the lung surface. Our data suggests that Ligustrazine may inhibit tumor metastasis, at least in part, through its anti-angiogenic activity.