Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 µmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

Hispidulin inhibits hepatocellular carcinoma growth and metastasis through AMPK and ERK signaling mediated activation of PPARγ.

Hispidulin, a phenolic flavonoid, exerts potent cytotoxicity towards a variety of human cancers. However, the effects of hispidulin on hepatocellular carcinoma (HCC) and underlying molecular mechanisms of its action remain elusive. The present study investigated the effect of hispidulin on HCC in experimental models, including tumor cell lines and mouse tumor xenograft. Results demonstrated that hispidulin was cytotoxic and anti-proliferative to HCC cell lines (SMMC7721 and Bel7402). Hispidulin activated caspase-3 and triggered apoptosis in HCC cells. Moreover, hispidulin inhibited cell migration and invasion by inhibiting the expression of matrix metalloproteinases (MMP-2, MMP-9) and by inducing tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. Hispidulin activated peroxisome proliferator-activated receptor γ (PPARγ) signaling which mainly contributed to its cytotoxicity in HCC cells. Remarkably, GW9662 (a PPARγ inhibitor) or PPARγ targeting siRNA significantly abrogated the anti-proliferative, pro-apoptotic, and anti-metastatic effects of hispidulin in HCC cells. Furthermore, hispidulin induced activation of PPARγ which was associated with increased phosphorylation of AMPK, ERK, JNK in HCC cells. Compound C (an AMPK inhibitor) or PD98059 (a MEK inhibitor) partly reversed the effects of hispidulin on PPARγ signaling in HCC cells. In contrast, no significant changes in PPARγ signaling were observed in HCC cells pretreated with SP600125 (a JNK inhibitor), while SP6000125 significantly inhibited the anti-cancer effects of hispidulin in HCC cells. Hispidulin administration effectively suppressed Bel7402 xenograft tumor growth and lung metastasis in vivo. Our findings indicate that PPARγ activation by hispidulin effectively suppressed HCC cell growth and metastasis both in vitro and in vivo.

Tegillarca granosa extract Haishengsu inhibits tumor activity via a mitochondrial­mediated apoptotic pathway.

Haishengsu (HSS) is an active natural extract isolated from Tegillarca granosa, which has previously been demonstrated to inhibit the proliferation of several types of cancer cells in vitro. Our previous study indicated that HSS may induce apoptosis to suppress growth of human hepatocellular carcinoma BEL­7402 cells by activating Fas pathway. The present study demonstrated that HSS treatment induces the in vitro apoptosis of BEL­7402 cells via the mitochondrial­mediated apoptotic pathway detected by DNA fragmentation assay, caspase activity assay and transmission electron microscopy assay, and inhibits tumor xenograft growth in vivo. Alterations in apoptotic regulatory proteins were detected, including decreased expression of B­cell lymphoma2 (Bcl­2), upregulation of Bcl­2­associated X protein and mitochondrial cytochrome c release, and downstream activation of apoptotic signaling. Furthermore, apoptotic induction was caspase­dependent, as indicated by cleavage of the caspase substrate, poly (ADP­ribose) polymerase. Oral administration of 62.5­250 mg/kg HSS markedly educed the growth of hepatocellular carcinoma tumor xenografts in nude mice. In addition, immunohistochemical staining for caspase­3 protein and transmission electron microscopy further indicated the induction of apoptosis in these tumor tissues. Taken together, the present study demonstrated that HSS may effectively induce apoptosis to suppress the growth of BEL­7402 cells in vitro and in vivo, and therefore may hold promise for further development as a novel cancer therapy.

Association between IL-4 and IL-4R Polymorphisms and Periodontitis: A Meta-Analysis.

Background. Previous studies have revealed that gene polymorphisms of inflammatory factors may influence the development or progression of periodontitis, a main cause of tooth loss in adults; however, due to limitations of individual studies, inconsistent findings were reported. Objective. To meta-analytically investigate the relationship between periodontitis and the Interleukin-4 (IL-4) and Interleukin-4 receptor (IL-4R) gene polymorphisms. Methods. Databases were searched for relevant case-control studies. After study selection based on the predefined selection criteria, methodological quality assessment and data extraction were conducted independently by two reviewers, before subsequent statistical analyses. Results. 37 studies involving 4,385 patients and 5,168 controls were included. All the studied IL-4 polymorphisms were not significantly associated with periodontitis, except the -33C/T (CT versus CC: OR = 0.50, 95% CI = 0.28-0.88) associated with reduced AgP susceptibility. Positive association was found between IL-4R Q551 polymorphism and periodontitis susceptibility in three genetic models (R versus Q: OR = 1.59, 95% CI = 1.14-2.22; QR versus QQ: OR = 1.84, 95% CI = 1.21-2.80; RR + QR versus QQ: OR = 1.82, 95% CI = 1.22-2.72). Conclusions. A positive association exists between the IL-4R Q551R polymorphism and occurrence of CP. The IL-4 -33 CT genotype is negatively associated with the occurrence of AgP.

Physcion inhibits the metastatic potential of human colorectal cancer SW620 cells in vitro by suppressing the transcription factor SOX2.

AIM: Physcion, an anthraquinone derivative, exhibits hepatoprotective, anti-inflammatory, anti-microbial and anti-cancer activities. In this study we examined whether and how physcion inhibited metastatic potential of human colorectal cancer cells in vitro. METHODS: Human colorectal cancer cell line SW620 was tested. Cell migration and invasion were assessed using a wound healing and Transwell assay, respectively. The expression levels of transcription factor SOX2 in the cells were modulated with shRNA targeting SOX2 and SOX2 overexpressing plasmid. The expression of target molecules involved in epithelial-mesenchymal transition (EMT) process and the signaling pathways was determined with Western blots or qRT-PCR. ROS levels were measured using DCF-DA. RESULTS: Physcion (2.5, 5 mol/L) did not affect the cell viability, but dose-dependently inhibited the cell adhesion, migration and invasion. Physcion also inhibited the EMT process in the cells, as evidenced by the increased epithelial marker E-cadherin expression, and by decreased expression of mesenchymal markers N-cadherin, vimentin, fibronectin and α-SMA, as well as transcriptional repressors Snail, Slug and Twist. Physcion suppressed the expression of SOX2, whereas overexpression of SOX2 abrogated the inhibition of physcion on metastatic behaviors. Physcion markedly increased ROS production and phosphorylation of AMPK and GSK3ß in the cells, whereas the AMPK inhibitor compound C or the ROS inhibitor NAC abolished the inhibition of physcion on metastatic behaviors. CONCLUSION: Physcion inhibits the metastatic potential of human colorectal cancer cells in vitro via activating ROS/AMPK/GSK3ß signaling pathways and suppressing SOX2.

Purple sweet potato pigments scavenge ROS, reduce p53 and modulate Bcl-2/Bax to inhibit irradiation-induced apoptosis in murine thymocytes.

AIMS: Purple sweet potato (PSP) pigments were proved to protect murine thymocytes from (60)Co γ-ray-induced mitochondria-mediated apoptosis in our previous study. In this study, we further investigated the effect of PSP pigments on apoptosis related ROS, p53 and Bcl-2 family. METHODS: Cell viability was analyzed by MTT. Apoptosis was certified by DNA ladder detection. Reactive oxygen species (ROS) were detected using 2',7',- dichlorofluorescein diacetate (DCFH-DA) probe. P53, Bcl-2 and Bax proteins were analyzed by western blot. The activities of caspase-3 and caspase-9 were determined by fluorogenic substrates detection. RESULTS: PSP pigments treatment prior to 4Gy (60)Co γ-ray irradiation increased the cell viability and decrease the apoptosis. In the presence of PSP pigments, ROS was scavenged and followed by a p53-depression. A shift in Bcl-2/Bax ratio towards anti-apoptosis was observed as a result of p53-depression. The activities of caspase-9 and caspase-3 were reduced by PSP pigments pretreatment. CONCLUSIONS: PSP pigments have a cytoprotective activity against γ radiation. The protective effect of PSP pigments may be involving ROS scavenging, p53 depression and Bcl-2/Bax modulation in a caspase-dependent mitochondrial way.

Effects of the novel vascular targeting agent MDS-11P on tumor vascularity and its antitumor activity.

Vascular disrupting agents show selective effects on tumor established vasculature, and achieve encouraging results in both pre-clinical and clinical experiments. In the present study, we investigated the effects of a new CA4 derivative MDS-11 and its prodrug MDS-11P on vascular disrupting activity in vitro and in vivo. Surface plasmon resonance (SPR) and tubulin polymerization assay showed that MDS-11 interacted with tubulin directly and inhibited tubulin polymerization in a cell free system, and western blot assay further confirmed the action in the cellular level. MDS-11 was found to significantly disrupt the microtubulin skeleton in proliferating HUVECs than quiescent ones determined by confocal microscopy. Furthermore, MDS-11 was found to damage the HUVEC-formed tube quickly, but did not influence structures of microvessels from aortic ring possessing pericytes and smooth muscle cells until 3 h treatment. In A549 xenograft mice, immunohistochemistry staining of tumor sections revealed that a single dose of MDS-11P led to large areas of necrosis within tumor and reduced the number of tumor vessels, which was consolidated by perfused vascular volume assay. Pharmacokinetic studies of MDS-11P indicated that MDS-11P rapidly converted to the active form, MDS-11, and exhibited a much faster elimination in mice. The antitumor analysis using H22 and A549 mice xenograft models revealed that the growth inhibition rates of MDS-11P at 50 mg/kg (twice a day for three weeks) reached 59.4%, 60.5% respectively without obvious weight loss. Taken together, these results suggest that MDS-11 is a potential vascular disrupting agent for further development of antitumor drug.

Effects of Ginkgo biloba extract EGb761 on human colon adenocarcinoma cells.

AIMS: To investigate the effect of Ginkgo biloba extract (EGb761) on cell proliferation and apoptosis in human colon cancer cells. METHODS: Human colon cancer cell lines (HT-29) were cultured and incubated with various concentrations (0-320 mg/l) of EGb 761 solution for up to 72 h. Cell viability, cell apoptosis, cell cycle, expression of caspase-3, the mRNA levels of p53, and Bcl-2 were assessed. RESULTS: EGb 761 inhibited the growth of HT-29 cells in a time-dose-dependent manner. At 80 and 320 mg/L, EGb 761 increased the number of cells in the G0/G1 phase and reduced cells in the G2/M and S phase. EGb 761 treatment also increased the apoptosis ratio of the HT-29 cells. EGb 761 treatment was associated with an increase in caspase-3 activities, reduction in bcl-2 mRNA expression and elevation in p53 mRNA expression. CONCLUSION: EGb 761 inhibits the progression of human colon cancer cells. Its therapeutic effect may be related to enhanced caspase-3 activities, up-regulation of p53 and down-regulation of bcl-2 genes.

[Effect between felodipine plus irbesartan and felodipine plus metoprolol regimen on the sexual function in young and middle-aged women with hypertension].

OBJECTIVE: To compare the effects between felodipine plus irbesartan and felodipine plus metoprolol regimen on blood pressure and the sexual function in young and middle-aged hypertensive women. METHODS: In this prospective, randomized, parallelized, controlled and fixed combined therapy trial, 99 female patients (aged 18 to 60) with grade 1 and grade 2 hypertension (BP ≥ 140/90 mm Hg and < 179/109 mm Hg, 1 mm Hg = 0.133 kPa) were assigned to felodipine 5 mg q.d + irbesartan 150 mg q.d (F + I group, n = 49) and felodipine 5 mg q.d + metoprolol 47.5 mg q.d (F + M group, n = 50) group. Target blood pressure was < 140/90 mm Hg. The female sexual function index (FSFI) questionnaire, levels of serum estradiol and testosterone were assessed. Female sexual dysfunction was defined as a FSFI score of less than 25.5. Patients were followed up for 24 weeks. RESULTS: The rate of achieving blood pressure goal between 2 groups was similar at the 4th, 8th, 12th and 24th weeks respectively (42.9% vs. 62.0% at 4th week, 89.8% vs. 90.0% at 8th week, 93.9% vs. 94.0% at 12th week, 98.0% vs. 96.0% at 24th week, P > 0.05). Compared to baseline, scores for the items related to "desire" and "arousal" were significantly improved (P < 0.05), the level of the serum estradiol was significantly elevated [(50.3 ± 37.4) pg/L vs. (54.4 ± 10.8) pg/L before menopause, (18.4 ± 2.9) pg/L vs. (20.2 ± 3.1)pg/L after menopause, P < 0.05] and the level of the serum testosterone was significantly decreased [(722.8 ± 277.1) ng/L vs. (650.0 ± 156.0) ng/L before menopause, (841.2 ± 279.3) ng/L vs. (761.9 ± 197.8) ng/L after menopause, P < 0.05] in the F + I group, while scores for the items related to "sexual desire" and "lubrication" were statistically reduced (P < 0.01), the concentration of the serum estradiol was significantly reduced [(57.4 ± 9.7) pg/L vs. (51.1 ± 12.1) pg/L before menopause, (19.8 ± 2.3) pg/L vs. (17.8 ± 3.3) pg/L after menopause, P < 0.01] and the level of the serum testosterone was significantly increased [(775.6 ± 217.8) ng/L vs. (886.0 ± 186.4) ng/L before menopause, (812.5 ± 311.3) ng/L vs. (914.4 ± 300.2) ng/L after menopause, P < 0.01] in the F + M group. FSFI score was negatively correlated with age and systolic blood pressure levels. CONCLUSION: felodipine plus irbesartan or metoprolol for 24 weeks equally reduced blood pressure and the former regimen is superior to the latter on sexual function improvement in this patient cohort.

Polypeptide from Chlamys farreri attenuates murine thymocytes damage induced by ultraviolet B.

AIM: Polypeptide from Chlamys farreri (PCF, molecular mass is 879) is a new marine polypeptide compound isolated from Chlamys farreri. This study investigates the possible protective roles and the mechanism of PCF against ultraviolet B (UVB)-induced apoptosis in murine thymocytes. METHODS: The rate of apoptosis and caspase-3 activation was measured by flow cytometry. The expression of stress-response genes c-fos and c-jun was observed by RT-PCR. Western blot analysis was performed to determine the release of cytochrome c. RESULTS: It was found that UVB induced murine thymocyte death. The cells treated with UVB showed an increase in cytochrome c release, caspase-3 activity, as well as in the expression of c-fos and c-jun. In addition, all were involved in UVB-induced cell apoptosis. CONCLUSION: Our present observations pointed to the ability of PCF to avert UVB-induced apoptosis in thymocytes by modulating c-fos and c-jun expression, cytochrome c release, and the consequent activation of caspase-3, which were essential components of the UV-induced cell apoptotic pathway. The results suggested that PCF is a promising protective substance against UV radiation.

Molecular mechanisms of polypeptide from Chlamys farreri protecting HaCaT cells from apoptosis induced by UVA plus UVB.

AIM: To investigate the mechanism of polypeptide from Chlamys farreri (PCF) protecting HaCaT cells from apoptosis induced by UVA plus UVB in vitro. METHODS: An apoptotic model of UV irradiation-induced HaCaT cells was established. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, agarose gel electrophoresis, biochemical methods, and Western blotting were employed in the study. RESULTS: PCF inhibited the UV irradiation-induced apoptosis of HaCaT cells. PCF strongly reduced the intracellular reactive oxygen species level, enhanced activities of superoxide dismutase and glutathione peroxidase and increased the total anti-oxidative capacity in HaCaT cells following UV irradiation. Furthermore, we found that PCF could inhibit the phosphorylation of c-Jun amino-terminal kinase and the activity of caspase-3 in a concentration-dependent manner. CONCLUSION: PCF protected HaCaT cells from apoptosis induced by UVA plus UVB, mainly through decreasing the intracellular ROS level and increasing the activities of anti-oxidative enzymes to block the ROS-JNK-caspase-3-apoptosis signaling pathway.

Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from Chlamys farreri in human HaCaT keratinocytes.

Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes, including apoptosis. Polypeptide from Chlamys farreri (PCF) is a novel marine active material isolated from the gonochoric Chinese scallop C. farreri. In our previous studies, PCF was found to be an effective antioxidant inhibiting UVA-induced ROS production and a potential inhibitory agent for UVA-induced apoptosis in the human keratinocyte cell line HaCaT. The intracellular mechanisms of how PCF protects HaCaT cells from UVA-induced apoptosis are not understood. Thus, we here investigate the effect of PCF on UVA-induced intracellular signaling of apoptosis. Pretreatment with the ROS scavenger N-acetylcysteine (NAC), the p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor Ac-DEVD-CHO was found to effectively prevent UVA-induced apoptosis, indicating that ROS, p38 MAPK and caspase-3 play important roles in apoptosis. H(2)O(2)-induced apoptosis was attenuated by PCF, suggesting that PCF plays its anti-apoptotic role through its antioxidant activity. In addition, PCF treatment inhibited UVA-induced p38 MAPK activation and caspase-3 activation, as assayed by Western blot analysis and flow cytometry, respectively. Our results suggest that PCF attenuates UVA-induced apoptosis through a reduction of ROS generation and diminished p38 MAPK and caspase-3 activation.

Acidic oligosaccharide sugar chain, a marine-derived oligosaccharide, activates human glial cell line-derived neurotrophic factor signaling.

Gial derived neurotrophic factor (GDNF) modulates neuronal cell differentiation during development and protects against neurodegeneration by preventing apoptosis at maturity. GDNF's role in tissue maintenance has generated interest in the therapeutic potential of GDNF in treating neurological disorders such as Parkinson's disease. Heparan sulfate has been shown to be essential for GDNF signaling and altering the levels of heparan sulfate promotes or inhibits GDNF functional activity. To search for other oligosaccharides capable of modulating GDNF activity as potential therapeutic molecules, we investigated the effect of acidic oligosaccharide sugar chain (AOSC) and its sulfated derivative on GDNF induced neurotrophic events by using Western-blotting, immunofluorescence cell staining, and immunoprecipitation techniques in PC12 cells expressing the GDNF receptors GFR alpha 1-Ret. AOSC significantly improved the neurite outgrowth and activated c-Ret phosphorylation in PC12-GFR alpha 1-Ret cells, but its sulfated derivative inhibited GDNF activity. Studies to understand the opposing biological effects of AOSC and its sulfated derivative on GDNF activity demonstrated that reduced GDNF binding to PC12-GFR alpha 1-Ret cell surface in the presence of the sulfated derivative likely suppressed GDNF activity as both AOSC and its sulfated derivatives had similar binding affinities to GDNF. This study illustrates the importance of oligosaccharide structure and charge on influencing GDNF activity and the potential use of oligosaccharides in modulating GDNF activity for therapeutic purposes.

[Inhibition of postharvest decay and induction of defensive enzymes by pure oxygen in Chinese bayberry fruit].

Chinese bayberry fruits were stored in air (control) or pure oxygen atmosphere for up to 12 days at 5 degrees C to investigate the effects of high oxygen on decay control and its relation to the induction of defensive enzyme activities. The results showed that exposure of Chinese bayberry to pure oxygen significantly prevented fruit decay. At the end of the storage period, the decay index of fruits exposed to pure oxygen was only 17% while that of control fruits reached 54% (Fig.1). Pure oxygen caused a significant increase in chitinase and beta-1,3-glucanase activities which reached a peak on the 6th day of storage (Fig.2). Phenylalanine ammonium-lyase (Fig.3A) and peroxidase (Fig.4) activities as well as total phenolic content (Fig.3B) increased more quickly and stayed at significantly higher levels in fruits exposed to pure oxygen during storage than the control fruits. These results suggest that the inhibition of postharvest fruit decay by high oxygen was related to the induction of defensive enzyme activities. The induced disease resistance may be involved in the mechanisms by which high oxygen treatment inhibited fruit decay in Chinese bayberry.