CD147 Sparks Atherosclerosis by Driving M1 Phenotype and Impairing Efferocytosis.

BACKGROUND: Atherosclerosis is a globally prevalent chronic inflammatory disease with high morbidity and mortality. The development of atherosclerotic lesions is determined by macrophages. This study aimed to investigate the specific role of myeloid-derived CD147 (cluster of differentiation 147) in atherosclerosis and its translational significance. METHODS AND RESULTS: We generated mice with a myeloid-specific knockout of CD147 and mice with restricted CD147 overexpression, both in an apoE-deficient (ApoE-/-) background. Here, the myeloid-specific deletion of CD147 ameliorated atherosclerosis and inflammation. Consistent with our in vivo data, macrophages isolated from myeloid-specific CD147 knockout mice exhibited a phenotype shift from proinflammatory to anti-inflammatory macrophage polarization in response to lipopolysaccharide/IFN (interferon)-γ. These macrophages demonstrated a weakened proinflammatory macrophage phenotype, characterized by reduced production of NO and reactive nitrogen species derived from iNOS (inducible NO synthase). Mechanistically, the TRAF6 (tumor necrosis factor receptor-associated factor 6)-IKK (inhibitor of κB kinase)-IRF5 (IFN regulatory factor 5) signaling pathway was essential for the effect of CD147 on proinflammatory responses. Consistent with the reduced size of the necrotic core, myeloid-specific CD147 deficiency diminished the susceptibility of iNOS-mediated late apoptosis, accompanied by enhanced efferocytotic capacity mediated by increased secretion of GAS6 (growth arrest-specific 6) in proinflammatory macrophages. These findings were consistent in a mouse model with myeloid-restricted overexpression of CD147. Furthermore, we developed a new atherosclerosis model in ApoE-/- mice with humanized CD147 transgenic expression and demonstrated that the administration of an anti-human CD147 antibody effectively suppressed atherosclerosis by targeting inflammation and efferocytosis. CONCLUSIONS: Myeloid CD147 plays a crucial role in the growth of plaques by promoting inflammation in a TRAF6-IKK-IRF5-dependent manner and inhibiting efferocytosis by suppressing GAS6 during proinflammatory conditions. Consequently, the use of anti-human CD147 antibodies presents a complementary therapeutic approach to the existing lipid-lowering strategies for treating atherosclerotic diseases.

Endovascular embolization for basal ganglia and thalamic arteriovenous malformations.

Background: Basal ganglia and thalamic arteriovenous malformations (AVMs) represent a special subset of malformations. Due to the involvement of vital brain structures and the specifically fine and delicate angioarchitecture of these lesions, it presents unique therapeutic challenges and technical difficulties that require thorough treatment planning, individualized treatment strategies, and advanced techniques for good clinical outcome. Method: In this study, we presented a series of ruptured basal ganglia and thalamic AVMs embolized via a transarterial, transvenous or combined approach. Herein, we summarized our treatment experience and clinical outcomes to further evaluate the effectiveness and safety of endovascular embolization for these AVMs as well as the indications, therapy strategies, and techniques of embolization procedures. Results: Twelve patients with basal ganglia and thalamus AVMs were included in the study. Their average age was 23.83 ± 16.51 years (range, 4-57 years) with a female predominance of 67% at presentation. The AVMs were located in the thalamus in 3 (25%) patients, in the basal ganglia in 3 (25%) patients, and in both sites of the brain in 6 (50%) patients. There were 5 AVMs located on the left side and 7 on the right. The mean nidus diameter was 3.32 ± 1.43 cm (range 1.3-6.1 cm). According to the Spetzler-Martin grading classification, 4 (33.3%) brain AVMs were Grade III, 7 (58.3%) were Grade IV, and 1 (8.3%) was Grade V. All of them presented with bleeding at admission: four of these patients presented with an intracerebral hemorrhage (ICH), 8 ICH in combination with intraventricular hemorrhage (IVH), and no patient with subarachnoid hemorrhage (SAH). Among these patients treated with endovascular embolization, 7 patients were treated by the transarterial approach, 4 patients transvenous approach, and 1 patient underwent the combined approach. A single embolization procedure was performed in 6 patients (50%) and the other 6 cases (50%) were treated in a staged manner with up to three procedures. Procedure-related complications occurred only in two patient (16.7%). Complete AVM obliteration was obtained in 7 patients (58.3%), and partial obliteration was in 4 patients (33.3%). Overall, good or excellent outcomes were obtained in 7 patients (58.3%), and poor functional outcome was observed in 5 patients (41.7%) at the last follow-up. All survived patients achieved anatomic stabilization and there was no postoperative bleeding or recurrence in the follow-up. Conclusion: The management of the basal ganglia and thalamic AVMs is a great challenge, which needs multimodal individualized treatment to improve the chances of radiographic cure and good outcomes. Endovascular therapy is safe and effective in the treatment of cerebral AVMs particularly for deep-seated AVMs such as the basal ganglia and thalamus. Our results demonstrate a high rate of anatomic obliteration with an acceptable rate of complications in the endovascular treatment of these vasculopathies via a transarterial approach or a transvenous approach.

Computed Tomography Features and Clinical Prognostic Characteristics of Hepatoid Adenocarcinoma of the Stomach.

PURPOSE: Hepatoid adenocarcinoma of the stomach (HAS) is a highly malignant and aggressive tumor. The purpose of this study was to describe the clinical, computed tomography (CT), and prognostic features of HAS to increase the awareness of this entity and determine its distinguishing features from non-HAS tumors. METHODS: The CT features and clinical data of 47 patients in our hospital with pathologically documented HAS were retrospectively analyzed, and the relevant differences between pure HAS (pHAS) and mixed HAS (mHAS) were determined. In addition, 141 patients with non-HAS tumors in the same T stage in the same period were selected as the control group. The data were compared between the two groups, and factors affecting the prognosis of HAS were analyzed. In addition, we included 9 patients with HAS and 27 patients with non-HAS tumors from another center for external validation. RESULTS: The patients in the HAS group were predominantly men (n = 33), and the tumor location was mostly the cardia or fundus (n = 27). Between the HAS and non-HAS groups, there were observed differences in terms of: sex, serum alpha-fetoprotein (AFP), carbohydrate antigen (CA)-125, and CA-724 levels; longest tumor diameter; degree of differentiation; vascular invasion; N stage, M stage, and tumor-node-metastasis (TNM) stage; thickest tumor diameter; plain CT attenuation; arterial-phase CT attenuation; CT attenuation between the venous and arterial phases; enhancement modes; and degrees of enhancement (all P < 0.05). In the data from another center for external validation, there were observed differences in terms of: age, degree of differentiation, vascular invasion, thickest tumor diameter, the ratio of arterial CT attenuation to CT attenuation of the abdominal aorta at the same level (RA), CT attenuation difference between the venous phase and arterial phase (HUv-a) (all P < 0.05). The results of the multivariate analysis revealed that the independent factors for differentiation were serum AFP level (P = 0.001), M stage (P = 0.038), and tumor enhancement on CT (P = 0.014). Among patients in the HAS group, 72.34% had pHAS and 27.66% had mHAS. The thickest tumor diameter and the longest short diameter of the metastatic lymph nodes of the mHAS group were on average 6.39 cm and 1.45 cm, respectively, which were larger than those in the pHAS group. The median progression-free survival time was 18.25 months in the HAS group, which was shorter than that in the non-HAS group (72.96 months; P = 0.001). The median overall survival time in the HAS group was 24.80 months, which was shorter than that in the non-HAS group (67.96 months; P = 0.001). The factors affecting the prognosis of HAS were M stage (P = 0.001), overall TNM stage (P = 0.048), presence of vascular cancer emboli (P = 0.040), and pHAS type (P = 0.046). Multifactorial analysis revealed that M stage (P = 0.027) and pHAS type (P = 0.009) were independent risk factors affecting the prognosis of HAS. CONCLUSION: Although HAS is a rare clinical entity, it should be considered in the differential diagnosis of gastric tumors. Patients with HAS often have advanced-stage disease at presentation and a worse prognosis than patients with non-HAS tumors. CT findings, combined with laboratory results, can support the diagnosis of HAS. However, the final diagnosis needs to be confirmed with a histopathologic examination. If the postoperative pathologic findings reveal the mHAS type, a rapid clinical intervention and a detailed follow-up with CT are essential.

Intracranial solitary fibrous tumors: A report of two cases and a review of the literature.

Solitary fibrous tumors (SFTs) are uncommon, with the pleura as a site of predilection. Central nervous system SFTs, particularly intracranial SFTs, are extremely rare. The lesions are generally benign and localized, and surgery is the main therapeutic solution. The current study reports the cases of a patient who presented with right haunch pain, right leg weakness and paresthesias for several months, and a patient with a history of unexpected loss of consciousness. Magnetic resonance imaging revealed the presence of lesions, with a spindle cell morphology evident on pathological examination. The immunohistochemical examination demonstrated a strong immunoreaction for cluster of differentiation 34, which supported the diagnosis of an SFT. Following a near-total resection, the patients had a good neural prognosis. The present study also provides a literature review, discussing the imageological and pathological characteristics of SFT, and the diagnostic methods that aid in distinguishing the entity from other spindle-cell central nervous system tumors.

KRAS-related noncoding RNAs in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a poor overall prognosis. However, curative resection during the early stages of the disease can greatly improve survival rates, highlighting the importance of early screening and detection. Studies of noncoding RNAs, primarily microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), provide important insights into strategies for the early detection of KRAS-driven PDAC. Here, we summarize our studies and review current reports on research investigating KRAS-related miRNAs and lncRNAs, emphasizing their aberrant expression, mechanisms, carcinogenic effects, and prognostic and predictive capacities in PDAC.

Primary Intracranial Atypical Teratoid/Rhabdoid Tumors: A Clinicopathologic and Neuroradiologic Study.

Atypical teratoid/rhabdoid tumors are rare malignant pediatric brain tumors. This study was performed to characterize the clinicopathologic and neuroradiologic characteristics of atypical teratoid/rhabdoid tumors from 8 patients, including 5 male and 3 female infants (median age, 67 months). Neuroimaging revealed bulky masses of heterogeneous intensity with inhomogeneous enhancement. Three cases were infratentorial and 5 were supratentorial. Histopathologically, the tumors were predominantly composed of rhabdoid cells and undifferentiated small cells, mixed with some spindle or epithelial components. The tumors displayed striking polyphenotypic immunoreactivity, including varying degrees of positivity for vimentin, epithelial membrane antigen, smooth-muscle actin, cytokeratin, glial fibrillary acidic protein, neurofilament protein, synaptophysin, and CD99, and immunonegativity for desmin, placental alkaline phosphatase, and INI-1. The median survival duration was 9.5 months (range, 1-15 months) despite aggressive therapy. These results suggest that atypical teratoid/rhabdoid tumors display distinct clinicopathologic characteristics and indicate a poor prognosis. Immunohistochemistry facilitates the appropriate diagnosis of these tumors.

Salvianolic acid B attenuates toxin-induced neuronal damage via Nrf2-dependent glial cells-mediated protective activity in Parkinson's disease models.

Salvianolic acid B (SalB), a bioactive compound isolated from the plant-derived medicinal herb Danshen, has been shown to exert various anti-oxidative and anti-inflammatory activities in several neurological disorders. In this study, we sought to investigate the potential protective effects and associated molecular mechanisms of SalB in Parkinson's disease (PD) models. To determine the neuroprotective effects of SalB in vitro, MPP+- or lipopolysaccharide (LPS)-induced neuronal injury was achieved using primary cultures with different compositions of neurons, microglia and astrocytes. Our results showed that SalB reduced both LPS- and MPP+-induced toxicity of dopamine neurons in a dose-dependent manner. Additionally, SalB treatment inhibited the release of microglial pro-inflammatory cytokines and resulted in an increase in the expression and release of glial cell line-derived neurotrophic factor (GDNF) from astrocytes. Western blot analysis illustrated that SalB increased the expression and nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The knockdown of Nrf2 using specific small interfering RNA (siRNA) partially reversed the SalB-induced GDNF expression and anti-inflammatory activity. Moreover, SalB treatment significantly attenuated dopaminergic (DA) neuronal loss, inhibited neuroinflammation, increased GDNF expression and improved the neurological function in MPTP-treated mice. Collectively, these findings demonstrated that SalB protects DA neurons by an Nrf-2 -mediated dual action: reducing microglia activation-mediated neuroinflammation and inducing astrocyte activation-dependent GDNF expression. Importantly the present study also highlights critical roles of glial cells as targets for developing new strategies to alter the progression of neurodegenerative disorders.

Construction of chimeric E3s expression plasmids targeting oncoprotein ras.

OBJECTIVE: To construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown. METHODS: We chose the binding domain of Raf-1, PI3K, RalGDS, and the function domain of F-Box as well as the U-Box to construct the plasmids. Then used the double enzyme, PCR, and sequence to test the validity and integrity of the cloned nucleotide fragments. The expression efficiency of the plasmids in eukaryotic cells was detected by Western blot analysis. RESULTS: Five of 6 plasmids in this study expressed the corresponding fusion proteins in HEK293T cells, and (RBD+CRD)(Raf-1)- U-Box-pcDNA3.1 can knocked down the protein level of Ras in PANC-1 cells. CONCLUSIONS: We successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.

[Construction of a miR-23a-27a cluster expression plasmid: a preliminary study of its function].

OBJECTIVE: To construct a miR-23a-27a cluster expression plasmid and to explore the target genes and function of the cluster. METHODS: The pre-miR-23a-27a-pcDNA3.1, pre-miR-23a and pre-miR-27a plasmids were cloned by molecular biology method, and their expression efficiency was tested by dual luciferase reporter gene assay and real-time PCR. Several possible target genes of miR-23a and miR-27a were chosen using softwares and further tested by dual luciferase reporter gene assay. Finally, the function of miR-27a was analyzed in MCF-7 cell by Western blot and real-time PCR. RESULTS: miR-23a and miR-27a were transcribed from pre-miR-23a-27a-pcDNA3.1, pre-miR-23a and pre-miR-27a plasmids in HEK293T cells, and both influenced the MRE of Sprouty2 gene in pRL-TK vector, and only miR-27a influenced the 3'-untranslated regions (UTR) full length of Sprouty2 gene while miR-27a did not influence the 3'-UTR of Sprouty2 gene with the sited-mutation in the MRE. The protein expression level of Sprouty2 gene was altered after transfection of pre-miR-27a-pcDNA3.1 plasmid while the RNA level remained unchanged. CONCLUSION: Sprouty2 may be the functional target gene of miR-27a, and the construction of plasmids in the study may provide a fundamental basis for the further functional investigation of miR-23a and miR-27a.

VEGF upregulates Homer 1a gene expression via the mitogen-activated protein kinase cascade in cultured cortex neurons.

In alternative splice variants of Homer 1 transcripts, Homer 1a messenger RNA (mRNA) has been shown to be upregulated selectively and rapidly by neural stimulation and represents a member of the immediate early gene (IEG) family. In our study, Homer 1 variants were expressed in cultured neurons as investigated by RT-PCR and Western blot analysis. After stimulation of VEGF, neurons selectively upregulated Homer 1a mRNA via Flk-1. The induction of Homer 1a mRNA peaked at 2h and sustained to 8h, while no significant change was observed of Homer 1b/c mRNA levels. Inhibitor analysis as well as Western blot analysis has indicated that the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays an important role in VEGF-stimulated induction of Homer 1a mRNA. These results demonstrate that MAPK is a key mediator that links distinct extracellular VEGF stimuli to the transcriptional activation of Homer 1a mRNA.

Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro.

AIM: To elucidate the role of dickkopf3 (Dkk3) in human pancreatic cancer cell growth. METHODS: Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by real-time reverse transcription polymerase chain reaction (real-time RT-PCR), Western blotting and immunofluorescence. Methylation of the Dkk3 promoter sequence was examined by methylation-specific polymerase chain reaction (MSP) and Dkk3 mRNA expression was determined by real-time RT-PCR after 5-aza-2'-deoxycytidine (5-aza-dC) treatment. The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells. The expression of ß-catenin, phosphorylated extracellular signal-regulated protein kinases (pERK) and extracellular signal-regulated protein kinases (ERK) was also examined by real-time RT-PCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells. RESULTS: The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested. The Dkk3 promoter sequence was methylated in the MIA PaCa-2 and AsPC-1 cell lines, which showed reduced Dkk3 expression. These two cell lines, which initially had a methylated Dkk3 promoter, showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent, 5-aza-dC, treatment (P < 0.05 or P < 0.01). When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa-2 cells, the ability of cells to proliferate decreased (P < 0.01), and the expression of ß-catenin and pERK was downregulated (P < 0.01). Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmid-transfected cells. CONCLUSION: Our findings, for the first time, implicate Dkk3 as a tumor suppressor in human pancreatic cancer, through the downregulation of ß-catenin expression via the ERK-mediated pathway.

[Significance of chemokine receptor CXCR3 expression in breast cancer].

OBJECTIVE: To investigate the expression of CXCR3 and its association with clinicopathologic features in breast carcinoma. METHODS: The expression level of CXCR3 in 18 samples of breast cancer and corresponding normal tissues was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis. Immunohistochemistry was carried out to examine the expression of CXCR3 in 80 breast cancers, 20 fibroadenomas and 15 normal breast tissues. RESULTS: (1) RT-PCR and real-time RT-PCR analysis showed a higher level of CXCR3 in breast cancer tissues than that in the corresponding normal breast tissues (P < 0.05). (2) Immunohistochemistry analysis showed that the positive rate of CXCR3 in breast cancer tissues was significantly higher than that in fibroadenomas and the normal breast tissues (P < 0.05). The expression level of CXCR3 in the lymph node-positive group was higher than that in the lymph node-negative group (P < 0.05). The expression of CXCR3 was positively correlated with the number of lymph nodes involved by metastasis, tumor size and pTNM tumor stage (P < 0.05). CONCLUSIONS: Chemokine receptor CXCR3 was up-regulated in breast cancer, and was associated with the progression of breast cancer. CXCR3 might be a novel molecular marker to predict lymph node metastasis and prognosis of breast cancer.

The miR-217 microRNA functions as a potential tumor suppressor in pancreatic ductal adenocarcinoma by targeting KRAS.

Aberrantly expressed microRNA (miRNA) is frequently associated with a variety of cancers, including pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated the expression and possible role of miR-217 in PDAC. Data obtained by locked nucleic acid in situ hybridization and real-time quantitative polymerase chain reaction showed that miR-217 was downregulated in 76.2% (16/21) of PDAC tissues and in all tested PDAC cell lines when compared with the corresponding normal pancreatic tissue. Overexpression of miR-217 in PDAC cells inhibited tumor cell growth and anchorage-independent colony formation and miR-217 decreased tumor cell growth in nude mouse xenografts in vivo. Using in silico predictions, KRAS was defined as a potential direct target of miR-217. Data from the dual-luciferase reporter gene assay showed that KRAS was a direct target of miR-217. Upregulation of miR-217 could decrease KRAS protein levels and reduce the constitutive phosphorylation of downstream AKT. Downregulation of miR-217 expression in PDAC cells could increase cell anchorage-independent colony formation and KRAS protein levels. Furthermore, miR-217 expression was observed to be negatively correlated with KRAS protein expression in PDAC cell lines. We conclude that the frequently downregulated miR-217 can regulate KRAS and function as a tumor suppressor in PDAC. Therefore, miR-217 may serve as a useful therapeutic agent for miRNA-based PDAC therapy.

[Expression of Twist, E-cadherin and N-cadherin in breast carcinoma and their clinical significance].

OBJECTIVE: To investigate the expression of Twist, E-cadherin and N-cadherin in breast carcinoma tissue and to analyse their effects on the breast carcinoma differentiation, size, infiltration and metastasis of the breast carcinoma. METHODS: The expression of Twist, E-cadherin and N-cadherin in 56 cases of breast invasive ductal carcinoma, 38 cases of invasive lobular carcinoma, 41 cases of carcinoma in situ and 10 cases of normal breast tissue was detected using immunohistochemistry. RESULTS: (1) The expression rate of Twist in three types of breast carcinoma was 46.4% (26/56), 79.0% (30/38) and 26.8% (11/41) respectively, and the expression of Twist in invasive lobular carcinoma was significantly higher than that in invasive ductal carcinoma and carcinoma in situ (P = 0.002, P = 0.000). The expression rate of E-cadherin in three types of breast carcinoma was 78.6% (44/56), 29.0% (11/38) and 80.5% (33/41) respectively, and the expression of E-cadherin in invasive ductal carcinoma and carcinoma in situ was significantly higher than that in invasive lobular carcinoma (P = 0.000, P = 0.000). The expression rate of N-cadherin in three types of breast carcinoma was 53.6% (30/56), 68.4% (26/38) and 31.7% (13/41) respectively, and the expression of N-cadherin in invasive ductal carcinoma and invasive lobular carcinoma was significantly higher than that in carcinoma in situ (P = 0.033, P = 0.001). (2) In all the 135 cases, the expression of Twist was not correlated with that of E-cadherin (P = 0.005, Spearman correlation coefficient = -0.239), however, there was a positive correlation between the expression of Twist and N-cadherin and statistically significant(P = 0.000, Spearman correlation coefficient = 0.319). (3) In the invasive ductal carcinoma, the expression of N-cadherin in poorly-differentiated carcinoma was significantly higher than that of the moderately-or well-differentiated ones (P = 0.004). (4) In the invasive lobular carcinoma, the expression of Twist in cases with lymph node metastasis was significantly higher than that of cases without metastasis (P = 0.037). CONCLUSIONS: Twist, E-cadherin and N-cadherin have different expression patterns in the three kinds of breast carcinoma. The positive expression of Twist was correlated to lymph node metastasis in invasive lobular carcinoma and the positive expression of N-cadherin was correlated to cell the tissue differentiation in invasive ductal carcinoma. Detection of the expression of these biomarkers may provide a valuable reference for the study of breast carcinoma progression, metastasis and for the judgment of the biological behavior of the carcinoma.

[Utilization of targeting specific proteins for ubiquitination and degradation].

Ubiquitin-proteasome pathway (UPP) can degrade specific proteins in eukaryotic cells. Targeting specific proteins for ubiquitination and degradation technique, based the theory of UPP and using certain chimeric E3s or protein-targeting chimeric molecules, can specifically knock down the targeted proteins. It is a promising new method for exploring the functions of genes or proteins and, together with gene-silencing technique, may play an important role in basic and clinical research.

FTY720 induces apoptosis in hepatocellular carcinoma cells through activation of protein kinase C delta signaling.

This study was aimed at elucidating the mechanism by which FTY720, a synthetic sphingosine immunosuppressant, mediated antitumor effects in hepatocellular carcinoma (HCC) cells. The three HCC cell lines examined, Hep3B, Huh7, and PLC5, exhibited differential susceptibility to FTY720-mediated suppression of cell viability, with IC(50) values of 4.5, 6.3, and 11 mumol/L, respectively. Although FTY720 altered the phosphorylation state of protein kinase B and p38, our data refuted the role of these two signaling kinases in FTY720-mediated apoptosis. Evidence indicates that the antitumor effect of FTY720 was attributable to its ability to stimulate reactive oxygen species (ROS) production, which culminated in protein kinase C (PKC)delta activation and subsequent caspase-3-dependent apoptosis. We showed that FTY720 activated PKC delta through two distinct mechanisms: phosphorylation and caspase-3-dependent cleavage. Cotreatment with the caspase-3 inhibitor Z-VAD-FMK abrogated the effect of FTY720 on facilitating PKC delta proteolysis. Equally important, pharmacologic inhibition or shRNA-mediated knockdown of PKC delta protected FTY720-treated Huh7 cells from caspase-3 activation. Moreover, FTY720 induced ROS production to different extents among the three cell lines, in the order of Hep3B > Huh7 >> PLC5, which inversely correlated with the respective glutathione S-transferase pi expression levels. The low level of ROS generation might underlie the resistant phenotype of PLC5 cells to the apoptotic effects of FTY720. Blockade of ROS production by an NADPH oxidase inhibitor protected Huh7 cells from FTY720-induced PKC delta activation and caspase-3-dependent apoptosis. Together, this study provides a rationale to use FTY720 as a scaffold to develop potent PKC delta-activating agents for HCC therapy.

Knockdown of survivin gene by vector-based short hairpin RNA technique induces apoptosis and growth inhibition in Burkitt's lymphoma Raji cell line.

Survivin is the smallest member of mammalian IAP (inhibitor of apoptosis) family. It is ubiquitous during embryonic development but is not expressed in normal post-natal tissues, except the thymus, colonic epithelial cells and CD34+ hematopoietic stem cells. However, its expression is upregulated during neoplastic transformation in both solid organ and hematological malignancies, including leukemia and lymphoma. In this study, we used RNA interference with short hairpin RNA (shRNA) technique to inhibit survivin expression in a Burkitt's lymphoma cell line Raji and validated its effects on apoptosis and cell proliferation. A survivin-shRNA expression vector were constructed and introduced into Raji cells. Expression of survivin mRNA and protein was assessed by RT-PCR and western blot analysis. Apoptosis index of transfected cells was quantified by flow cytometry and cell proliferation was enumerated by trypan blue exclusion. In Raji cells treated with survivin-shRNA expression vector, survivin mRNA levels were significantly reduced by 67.14% (transient transfection) and 64.28% (stable transfection) respectively, compared with control-shRNA treated group and PBS treated group (p<0.05). The levels of survivin protein were significantly reduced by 62.50% (transient transfection) and 60.93% (stable transfection), compared with the two control groups (p<0.05). Apoptosis index was significantly increased during transient transfection and stable transfection, respectively 31.20+/-2.45% and 29.40+/-1.72% (p<0.05). Survivin-shRNA inhibited the proliferation of Raji cells of stable transfection. In conclusion, the vector-based survivin-shRNA can effectively reduce the expression of survivin gene and induce apoptosis and growth inhibition of transfected Raji cells. We suggest that survivin can be regarded as an ideal target for new anticancer intervention of NHL.