Activation of protein kinase C-α isoform in murine melanoma cells with high metastatic potential

Metastasis is a multistep process in which protein kinase C (PKC) appears to be significantly involved. We analysed the activity and expression of classical (, , ) and novel PKC isoforms in B16-F1 and B16-BL6 melanoma cells maintained under different culture conditions in vitro. We used high and low concentrations of tyrosine and phenylalanine in different media (DMEM or RPMI 1640 respectively) that affect the metastatic potential and also the proliferative capacity of the cells. We also tested a weakly metastatic amelanotic B78-H1 melanoma cell line which is unaffected by the different culture conditions. In both B16 melanoma cell lines activation of PKC (without increased expression) occurred under growth conditions permissive of metastasis (DMEM). In contrast, the weakly metastatic amelanotic B78-H1 cell line showed a substantial inactivation of this isoform in the two different culture media, suggesting a specific involvement of PKC in the metastatic process. Moreover, in B16 melanoma cells, novel PKC was activated under culture conditions which stimulated growth but not metastasis (RPMI 1640). In order to define the relationship between PKC activation and the metastatic process we also determined the release of cathepsin B. No correlation between PKC activity and cathepsin B release in either B16 melanoma cell lines could be demonstrated.     

人多发性骨髓瘤裸鼠皮下移植瘤模型的建立

目的:探索人多发性骨髓瘤裸鼠皮下移植瘤模型建立的方法。方法:Balb/c裸鼠进行3GyX线照射预处理后24h,于其皮下接种2×107人多发性骨髓瘤细胞RPMI8226,每2~3d观察裸鼠体重的变化、肿瘤生长的情况、裸鼠的生存时间等,待裸鼠濒死或死亡,或者超过观察时间仍未死亡时,处死裸鼠,取其皮下结节及器官进行病理检测,同时摘眼球取血行血清免疫固定电泳检测。结果:荷瘤裸鼠出现皮下结节的高峰为荷瘤后第2~3周,结节体积基本达到高峰为荷瘤后第5~6周;裸鼠死亡高峰出现在荷瘤后第7周,荷瘤小鼠中位生存时间为荷瘤后52d;裸鼠处死后血清中未检测到人源单克隆免疫球蛋白;皮下结节HE染色证实瘤组织为浆细胞来源,器官HE染色显示肝脏部分炎性损伤。结论:采用RPMI8226人骨髓瘤细胞于Balb/c裸鼠皮下接种(接种细胞数为2×107个细胞)造模的方法,具有操作过程较简单、技术要求低,观察肿瘤生长较为直观等优点;同时采用X线照射预处理的方法可降低裸鼠免疫力,明显提高模型的成瘤率;人多发性骨髓瘤细胞RPMI8226皮下接种于裸鼠,肿瘤生长过程中很难有人源单克隆免疫球蛋白分泌入血;荷瘤裸鼠出现肝脏的损伤,也许可作为今后抗肿瘤药物疗效研究的指标之一。     

Antiproliferative activity and standardization of Tecomella undulata bark extract on K562 cells

Ethnopharmacological relevance

The bark of Tecomella undulata is primarily used in the treatment of syphilis, painful swellings and cancer by traditional healers. Also, it is claimed to be useful in treating urinary discharges, enlargement of spleen, leucorrhoea, leukoderma, tumors, liver disorders, gonorrhea, gout and promotes wound healing in Indian traditional system of medicine.

Aim

To establish a scientific validation for the antitumor effects of Tecomella undulata bark and explore the mechanistic pathway in chronic myeloid leukemia cell line, K562. The study was further extended to standardize the extract using quercetin as biomarker.

Methods

Induction of apoptosis by chloroform extract of Tecomella undulata bark (CTUB) was determined by MTT, Annexin V and caspase activation assays. The cell cycle analysis was done by flow cytometer and nuclear staining by DAPI. The standardization of the extract was performed through reverse phase-HPLC method under PDA detection.

Result

Results clearly showed the induction of apoptosis by CTUB in K562 cells. The effect was found to be dose dependent, having IC₅₀ of 30 μg/ml with activation of FAS, FADD, caspase 8, caspase 3/7 and fragmentation of DNA. The bioactive CTUB was determined to possess 0.03% (w/w) of quercetin.

Conclusion

The investigation clearly demonstrated the potential antitumor effect of CTUB, thereby validating the traditional claim. Quercetin, known to have anticancer activity is being reported and quantified for the first time from the bark of Tecomella undulata.     

The effects of Morinda citrifolia L. (noni) on the immune system: its molecular mechanisms of action

The aim of this study was to investigate the mechanisms involved in the immunomodulatory effects of Morinda citrifolia L. (noni) in vitro and in vivo in mice. In vitro, Tahitian Noni Juice (TNJ) and Noni fruit juice concentrates (NFJC) (1, 5mg/mL) potently activate cannabinoid 2 (CB2), but inhibit cannabinoid 1 (CB1) receptors in a concentration-dependant manner. In vivo, oral administration of TNJ ad libitum for 16 days decreased the production of IL-4, but increased the production of IFN-gamma. These results suggest that noni modulates the immune system via activating of the CB2 receptors, and suppressing of the IL-4, but increasing the production of IFN-gamma cytokines. It may also exert beneficial immunomodulation effects in conditions involving inadequate immune responses.     

In vitro effects of active components of Polygonum Multiflorum Radix on enzymes involved in the lipid metabolism

Ethnopharmacology relevance

Raw and processed Polygoni Multiflori Radix (PMR and PMRP) are used in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD), hyperlipidemia or related diseases. In our previous research, 2, 3, 5, 4′-tetrahydroxy-stilbene-2-O-β-d-glucoside (TSG) displayed the most important role in the total cholesterol (TC) lowering effect among all the chemical constituents of Polygonum multiflorum. Emodin and physcion displayed more favorable triglyceride (TG) reducing effects than TSG. However, there are few researches focus on the approach and mechanism of how do Polygonum multiflorum exhibit good lipid regulation activity. The targeted sites of active substances of Polygonum multiflorum are still not clearly elucidated. This research pays close attention to how major chemical components of Polygonum multiflorum affect the TC and TG contents in liver cells.

Materials and methods

In this research, a sensitive, accurate and rapid in vitro model, steatosis hepatic L02 cell, was used to explore target sites of active chemical substances of Polygonum multiflorum for 48 h. Steatosis hepatic L02 cell was exposed to emodin, physcion and TSG, respectively. The contents of four key enzymes in the pathway of synthesis and decomposition of TC and TG were investigated after exposure. Meanwhile, the contents of lipid transfer protein were also tested. The diacylgycerol acyltransferase 1 (DGAT1) controlled the biosynthesis of TG from free fatty acids while 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) limited the biosynthesis of TC. Hepatic triglyceride lipase (HTGL) and cholesterol 7α-hydroxylase (CYP7A) played the key role in the lipolysis procedure of TG and TC.

Results

The synthesis of TC and TG in steatosis L02 cells were apparently increased in the model group compared to the control group. Intracellular contents of HMG-CoA reductase and DGAT1 increased 32.33% and 56.52%, while contents of CYP7A and HTGL decreased 21.61% and 47.37%. Emodin, physcion and TSG all showed down-regulation effects on HMG-CoA reductase, while up-regulation effects on CYP7A. The most remarkable effect on HMG-CoA reductase was found on emodin. Emodin could reduce the DGAT1 content from 438.44±4.51 pg/mL in model group to 192.55±9.85 pg/mL (100 μm). The content of HTGL in 300 μm physcion group was 3.15±0.15 U/mL, which was more significantly effective than the control, lovastatin and fenofibrate group.

Conclusions

TSG could raise the content of CYP7A and then promote the lipolysis of cholesterol. Moreover, TSG also showed the best LDL-reducing effect. Emodin could inhibit HMG-CoA reductase and DGAT1, which were key enzymes in the synthesis of TC and TG. Physcion increased the content of HTGL, and then could boost the lipolysis of triglyceride. At the same time, physcion showed the best VLDL-reducing effect. In view of the above conclusions, we contributed the lipid regulation activity to an overall synergy of TSG, emodin and physcion.     

维生素K_3和三氧化二砷诱导多发性骨髓瘤细胞凋亡的协同作用

目的：探讨三氧化二砷（As2O3）联合维生素K3对人多发性骨髓瘤细胞株RPMI8226细胞生长抑制及诱导凋亡作用机制。方法：采用四甲基噻唑氮蓝比色法检测As2O3联合VK3对RPMI8226细胞生长抑制作用、DNA电泳、流式细胞术Annexin V/PI标记法检测凋亡,观察As2O3联维生素K3对RPMI8226细胞的增殖抑制及促凋亡作用;同时通过RT-PCR检测VEGF表达来观察VK3、As2O3对RPMI8226细胞的凋亡诱导作用及两者是否有协同作用。结果：VK3和As2O3二者均可明显抑制RPMI8226细胞系的增殖,其作用机制可能通过降低Bcl-2mRNA的表达、诱导调亡,这种效应具有时间与剂量依赖性;二者间具有协同作用。经VK3和As2O3作用后的RPMI8226细胞出现凋亡细胞的特征;维生素K3（VK3）、As2O3单独及联用作用于RPMI8226细胞DNA凝胶电泳可见明显的梯形条带。与单用As2O3相比,As2O3联合VK3作用于RPMI8226细胞48h后,细胞凋亡率显著增加（P〈0.05）,G0/G1期细胞比例升高,S期细胞减少。RT-PCR检测表明RPMI8226细胞经VK3、As2O3单独及联合作用后血管内皮生长因子VEGF表达明显降低。结论：VK3、As2O3单用及联用对RPMI8226细胞有明显的增殖抑制作用和诱导凋亡作用,且具有一定的量效和时效关系。可能通过VEGF表达降低而起作用。VK3和As2O3在诱导RPMI8226细胞凋亡中具有明显的协同作用。     

人芽囊原虫在不同培养基中生长状况的观察

目的筛选培养人芽囊原虫的最适培养基。方法将同一株人芽囊原虫阳性粪便标本以2×105细胞/管接种至RPMI1640、199和LES培养基中,加入20%小牛血清及青、链霉素,pH值为7.5,放置厌氧罐中于37℃恒温培养,每24h计数,每6d转种1次。观察人芽囊原虫在3种培养基中的存活时间、虫体密度和虫体形态。结果人芽囊原虫在RPMI1640培养基中存活时间最长、虫体密度最高,虫体以空泡型多见;在LES培养基中存活时间最短、虫体密度最低,但虫体形态清晰、规则;在199培养基中存活时间和虫体密度均介于前两者之间。结论RPMI1640培养基适宜人芽囊原虫的生长繁殖,为人芽囊原虫体外培养的首选培养基;LES培养基中虫体形态清晰、规则,可用于人芽囊原虫的形态学研究;199培养基也可用于人芽囊原虫的体外培养,但不作为首选。     

The effect of pharmacological PI3Kγ inhibitor on eotaxin-induced human eosinophil functions

BackgroundAsthma is characterized by chronic inflammation caused by activation of immune cells including Th2 lymphocytes and eosinophils. Phosphoinositide 3-kinase (PI3K) γ deficient asthmatic mice did not develop lung eosinophilia, although the detailed mechanisms are not well known. A CC chemokine eotaxin (CCL11) plays a prominent role in developing eosinophilic inflammation through CCR3. In this study, we tested the roles of PI3Kγ in eotaxin-induced eosinophil functions using a pharmacological inhibitor.MethodHuman peripheral blood eosinophils were isolated by CD16-negative selection method. The effect of AS605240, synthetic PI3Kγ inhibitor on eotaxin-induced adhesion, chemotaxis, and degranulation were studied using intracellular adhesion molecule-1 (ICAM-1)-coated plates, Boyden chamber system, ELISA for eosinophil-derived neurotoxin (EDN) levels in the culture supernatant, respectively. CCR3 expression levels and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were assessed by flowcytometry. Involvement of PI3Kγ in spontaneous apoptosis was studied using flowcytometry.ResultsAlthough AS605240 did not affect the eosinophil spontaneous apoptosis, eotaxin-induced chemotaxis, adhesion to ICAM-1 coated plate, and EDN release were inhibited by AS605240. AS605240 also inhibited the eotaxin-induced ERK1/2 phosphorylation without down-regulation of surface CCR3 expression.ConclusionThese results indicate that PI3Kγ inhibitor attenuates eotaxin-induced eosinophil functions by suppressing the downstream signaling of CCR3 without significant cytotoxicity. PI3Kγ plays an important role in the development of eosinophilic inflammation and blockade of PI3Kγ might be a therapeutic strategy for treatment of eosinophil-related diseases including asthma.     

Multimodal imaging for a theranostic approach in a murine model of B-cell lymphoma with engineered nanoparticles

Nanoparticles (NPs) are a promising tool for in vivo multimodality imaging and theranostic applications. Hyaluronic acid (HA)-based NPs have numerous active groups that make them ideal as tumor-targeted carriers. The B-lymphoma neoplastic cells express on their surfaces a clone-specific immunoglobulin receptor (Ig-BCR). The peptide A20-36 (pA20-36) selectively binds to the Ig-BCR of A20 lymphoma cells. In this work, we demonstrated the ability of core-shell chitosan-HA-NPs decorated with pA20-36 to specifically target A20 cells and reduce the tumor burden in a murine xenograft model. We monitored tumor growth using high-frequency ultrasonography and demonstrated targeting specificity and kinetics of the NPs via in vivo fluorescent reflectance imaging. This result was also confirmed by ex vivo magnetic resonance imaging and confocal microscopy. In conclusion, we demonstrated the ability of NPs loaded with fluorescent and paramagnetic tracers to act as multimodal imaging contrast agents and hence as a non-toxic, highly specific theranostic system.