Long-term prognostic benefit of adjuvant chemotherapy for patients with hepatoid adenocarcinoma of the stomach after radical resection: A national multicenter study.

BACKGROUND: There is no consensus on whether adjuvant chemotherapy (AC) is effective for hepatoid adenocarcinoma of the stomach (HAS). The aim of this study was to investigate the relationship between AC and the long-term prognosis of patients with HAS. METHODS: The clinicopathological data of 239 patients with primary HAS who underwent radical surgery from April 1, 2004 to December 31, 2019 in 14 centers in China were retrospectively analyzed. Patients were divided into the AC group (127 patients) and the nonadjuvant chemotherapy (NAC) group (112 patients). RESULTS: Kaplan‒Meier (KM) analysis showed that there were no significant differences in the 1-year3-year overall survival rate (OS) and 1-year, 3-year recurrence-free survival rate (RFS) between the AC group and the NAC group (1-year OS: 85.6% vs. 79.8%, 3-year OS: 59.8% vs. 62.4%, 1-year RFS: 69.8% vs. 74.4%, 3-year RFS: 57.2% vs. 55.9%, all P > 0.05). The subpopulation treatment effect pattern plots (STEPP) did not show treatment heterogeneity of AC in patients with HAS. The proportions of local recurrence and metastasis sites in the two groups were similar. Although the smoothed hazard curves of the NAC and AC groups crossed, the peak hazard time was later in the AC group (5.9 and 4.7 months), and the peak hazard rate was lower (0.032 and 0.038, P = 0.987). CONCLUSION: The current AC regimen may not significantly improve the survival of patients with HAS after radical surgery.

Nomogram to Predict Recurrence and Guide a Pragmatic Surveillance Strategy After Resection of Hepatoid Adenocarcinoma of the Stomach: A Retrospective Multicenter Study.

BACKGROUND: An accurate recurrence risk assessment system and surveillance strategy for hepatoid adenocarcinoma of the stomach (HAS) remain poorly defined. This study aimed to develop a nomogram to predict postoperative recurrence of HAS and guide individually tailored surveillance strategies. METHODS: The study enrolled all patients with primary HAS who had undergone curative-intent resection at 14 institutions from 2004 to 2019. Clinicopathologic variables with statistical significance in the multivariate Cox regression were incorporated into a nomogram to build a recurrence predictive model. RESULTS: The nomogram of recurrence-free survival (RFS) based on independent prognostic factors, including age, preoperative carcinoembryonic antigen, number of examined lymph nodes, perineural invasion, and lymph node ratio, achieved a C-index of 0.723 (95% confidence interval [CI], 0.674-0.772) in the whole cohort, which was significantly higher than those of the eighth American Joint Committed on Cancer (AJCC) staging system (C-index, 0.629; 95% CI, 0.573-0.685; P < 0.001). The nomogram accurately stratified patients into low-, middle-, and high-risk groups of postoperative recurrence. The postoperative recurrence risk rates for patients in the middle- and high-risk groups were respectively 3 and 10 times higher than for the low-risk group. The patients in the middle- and high-risk groups showed more recurrence and metastasis, particularly multiple site metastasis, within 36 months after the operation than those in the low-risk group (low, 2.2%; middle, 8.6%; high, 24.0%; P = 0.003). CONCLUSIONS: The nomogram achieved good prediction of postoperative recurrence for the patients with HAS after radical resection. For the middle- and high-risk patients, more active surveillance and targeted examination methods should be adopted within 36 months after the operation, particularly for liver and multiple metastases.

Analysis of 20 patients with laparoscopic extended right colectomy.

BACKGROUND: Currently, the standard surgical procedure for right colon cancer is complete mesocolic excision. Whether preventive extended lymph node dissection for colon cancer located in the hepatic flexure or right transverse colon should be performed remains controversial because the safety and effectiveness of the operation have not been proven, and infrapyloric lymph nodes (No. 206) and lymph nodes in the greater curvature of the stomach (No. 204) have not been strictly defined and distinguished as surgical indicators in previous studies. AIM: To analyze the metastatic status of infrapyloric lymph nodes and lymph nodes of the greater curvature of the stomach and perioperative complications and systematically evaluate the feasibility and safety of laparoscopic extended right colectomy using prospective data collected retrospectively. METHODS: The study was a clinical study. Twenty patients with colon cancer who underwent laparoscopic extended right colon resection in our hospital from June 2020 to May 2021 were included. RESULTS: Among the patients who underwent extended right colon resection, there were no intraoperative complications or conversion to laparotomy; 2 patients had gastrocolic ligament lymph node metastasis, and 5 patients had postoperative complications. The patients with postoperative complications received conservative treatment. CONCLUSION: Laparoscopic extended right colon resection is safe. However, malignant tumors located in the liver flexure or the right-side transverse colon are more likely to metastasize to the gastrocolic ligament lymph nodes, and notably, the incidence of gastroparesis was high. The number of patients was small, and the follow-up time was short. It is necessary to further increase the sample size to evaluate the No. 204 and No. 206 lymph node metastasis rates and the long-term survival impact.

Nomogram for predicting chylous ascites after right colectomy.

BACKGROUND: Chylous ascites following right colectomy has a high incidence which is a critical challenge. At present, there are few studies on the factors affecting chylous ascites after right colectomy and especially after D3 Lymphadenectomy. A predictive model for chylous ascites has not yet been established. Therefore, we created the first nomogram to predict the incidence of chylous ascites after right hemicolectomy. AIM: To analyze the risk factors for chylous ascites after right colectomy and establish a nomogram to predict the incidence of chylous ascites. METHODS: We retrospectively collected patients who underwent right hemicolectomy between January 2012 and May 2021 and were pathologically diagnosed with cancer. Multivariate logistic regression was used to analyze the influencing factors of chylous ascites and a nomogram was established. The predictive ability was assessed by the area under the receiver operating characteristic (ROC) curve. RESULTS: Operative time, the type of operation (standard or extended), the number of lymph nodes retrieved, and somatostatin administration were considered important risk factors. Multivariate logistic regression and nomograms can be used to accurately predict whether chylous ascites occurs. The area under the ROC curve of the model is 0.770. The C-statistic of this model is 0.770 which indicates that it has a relatively moderate ability to predict the risk of chylous ascites. CONCLUSION: We found a novel set of risk factors, created a nomogram, and validated it. The nomogram had a relatively accurate forecasting ability for chylous ascites after right hemicolectomy and can be used as a reference for risk assessment of chylous ascites and whether to prevent it after surgery.

Assessment of Clinicopathological Characteristics and Development of an Individualized Prognostic Model for Patients With Hepatoid Adenocarcinoma of the Stomach.

Importance: Few studies have examined the clinicopathological characteristics and prognoses of patients with hepatoid adenocarcinoma of the stomach (HAS). Objective: To explore the clinicopathological characteristics and prognoses of patients with HAS and develop a nomogram to predict overall survival (OS). Design, Setting, and Participants: This prognostic study involved a retrospective analysis of data from 315 patients who received a diagnosis of primary HAS between April 1, 2004, and December 31, 2019, at 14 centers in China. Main Outcomes and Measures: OS and prognostic factors. Patients were randomly assigned to a derivation cohort (n = 220) and a validation cohort (n = 95). A nomogram was developed based on independent prognostic factors identified through a multivariable Cox mixed-effects model. Results: Among 315 patients with HAS (mean [SD] age, 61.9 [10.2] years; 240 men [76.2%]), 137 patients had simple HAS (defined as the presence of histologically contained hepatoid differentiation areas only), and 178 patients had mixed HAS (defined as the presence of hepatoid differentiation areas plus common adenocarcinoma areas). Patients with simple HAS had a higher median preoperative α-fetoprotein level than those with mixed HAS (195.9 ng/mL vs 48.9 ng/mL, respectively; P < .001) and a higher rate of preoperative liver metastasis (23 of 137 patients [16.8%] vs 11 of 178 patients [6.2%]; P = .003). The 3-year OS rates of patients with simple vs mixed HAS were comparable (56.0% vs 60.0%; log-rank P = .98). A multivariable Cox analysis of the derivation cohort found that the presence of perineural invasion (hazard ratio [HR], 2.13; 95% CI, 1.27-3.55; P = .009), preoperative carcinoembryonic antigen levels of 5 ng/mL or greater (HR, 1.72; 95% CI, 1.08-2.74; P = .03), and pathological node category 3b (HR, 3.72; 95% CI, 1.34-10.32; P = .01) were independent risk factors for worse OS. Based on these factors, a nomogram to predict postoperative OS was developed. The concordance indices of the nomogram (derivation cohort: 0.72 [95% CI, 0.66-0.78]; validation cohort: 0.72 [95% CI, 0.63-0.81]; whole cohort: 0.71 [95% CI, 0.66-0.76]) were higher than those derived using the American Joint Committee on Cancer's AJCC Cancer Staging Manual (8th edition) pathological tumor-node-metastasis (pTNM) staging system (derivation cohort: 0.63 [95% CI, 0.57-0.69]; validation cohort: 0.65 [95% CI, 0.56-0.75]; whole cohort: 0.64 [95% CI, 0.59-0.69]) and those derived using a clinical model that included pTNM stage and receipt of adjuvant chemotherapy (derivation cohort: 0.64 [95% CI, 0.58-0.69]; validation cohort: 0.65 [95% CI, 0.56-0.75]; whole cohort: 0.64 [95% CI, 0.59-0.69]). Based on the nomogram cutoff of 10 points, the whole cohort was divided into high-risk and low-risk groups. The 3-year OS rate of patients in the high-risk group was significantly lower than that of patients in the low-risk group (29.7% vs 75.9%, respectively; log-rank P < .001), and the 3-year prognosis of high-risk and low-risk groups could be further distinguished into pTNM stage I to II (33.3% vs 80.2%; exact log-rank P = .15), stage III (34.3% vs 71.3%; log-rank P < .001), and stage IV (15.5% vs 70.3%; log-rank P = .009). Conclusions and Relevance: This study found that perineural invasion, preoperative carcinoembryonic antigen levels of 5 ng/mL or greater, and pathological node category 3b were independent risk factors associated with worse OS. An individualized nomogram was developed to predict OS among patients with HAS. This nomogram had good prognostic value and may be useful as a supplement to the current American Joint Committee on Cancer TNM staging system.

Splenosis masquerading as gastric stromal tumor: A case report.

BACKGROUND: Splenosis is a rare benign disease that often disguises itself as a malignant tumor. There are few articles providing a comprehensive description of splenosis, especially cases located in the stomach being treated by laparoscopic surgery. CASE SUMMARY: A 44-year-old man presented with recurrent upper abdominal pain for more than half a year. The patient had splenic rupture caused by trauma more than 10 years ago and underwent splenectomy. An abdominal contrast-enhanced computed tomography scan revealed an irregular soft tissue density. Gastroscopy revealed an approximately 3.0 cm × 3.0 cm mucosal eminence at the posterior wall of the upper segment of the gastric body. Biopsy was not performed since the lesion was found under the mucosa and the gastric mucosa appeared normal. According to these findings, a diagnosis of gastric stromal tumor was made, although a definitive differential diagnosis was not known before surgery. When laparoscopic resection of the gastric stromal tumor was performed, an astonishing finding was made when postoperative pathology showed that the lesion comprised typical spleen tissue. CONCLUSION: This case highlights the strong similarities between splenosis and malignant tumors. A detailed medical history combined with various effective auxiliary examinations can help improve differential diagnosis.

Loss of BMI-1 dampens migration and EMT of colorectal cancer in inflammatory microenvironment through TLR4/MD-2/MyD88-mediated NF-κB signaling.

Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment created by immune cells facilitates tumor migration. Epithelial-mesenchymal transition (EMT) is involved in the progression of cancer invasion and metastasis in an inflammatory microenvironment. B-lymphoma Moloney murine leukemia virus insertion region 1 (BMI-1) acts as an oncogene in various tumors. Ectopic expression of Bmi-1 have an effect on EMT and invasiveness. The purpose of this study was to investigate the efficacy of BMI-1 on inflammation-induced tumor migration and EMT and the underlying mechanism. We observed that the expression of BMI-1, TNF-α, and IL-1ß was significantly increased in HT29 and HCT116 cells after THP-1 Conditioned-Medium (THP-1-CM) stimulation. Additionally, inhibition of BMI-1 impeded cell invasion induced by THP-1-CM-stimulation in both HT29 and HCT116 cells. BMI-1 knockdown remarkably repressed THP-1-CM-induced EMT by regulating the expression of EMT biomarkers with an increase in E-cadherin accompanied by decrease in N-cadherin and vimentin. Furthermore, downregulation of BMI-1 dramatically impeded THP-1-CM-triggered Toll-like receptor 4(TLR4)/myeloid differentiation protein 2(MD-2)/myeloid differentiation factor 88(MyD88) activity by repressing the expression of the TLR4/MD-2 complex and MyD88. Further data demonstrated that knockout of BMI-1 also dampened NF-κB THP-1-CM-triggered activity. Taken all data together, our findings established that BMI-1 modulated TLR4/MD-2/MyD88 complex-mediated NF-κB signaling involved in inflammation-induced cancer cells invasion and EMT, and therefore, could be a potential chemopreventive agent against inflammation-associated colorectal cancer. HIGHLIGHTS: Establishment of an inflammatory microenvironment. Suppression of BMI-1 reverses THP-1-CM-induced inflammatory cytokine production in CRC. Loss of BMI-1 suppressed TLR4/MD-2/MyD88 complex-mediated NF-κB signaling.

Inhibition of cerebral ischemia/reperfusion-induced injury by adenovirus expressed C-terminal amino acids of GluR6.

GluR6 kainate receptor subunit is largely expressed in hippocampus of brain regions and plays an important role in brain ischemia/reperfusion-mediated neuronal cell death. Our previous researches have shown that cerebral ischemia/reperfusion could facilitate the assembly of GluR6 and postsynaptic density protein 95(PSD95) as well as mixed lineage kinase 3(MLK3) and further induce the activation of c-Jun NH2-terminal kinase 3(JNK3), leading to neuronal death of hippocampal CA1. Here, we show that over-expression of C-terminal amino acids of GluR6 can interrupt the combination of GluR6 with PSD95, inhibit the assembly of GluR6.PSD-95.MLK3 signaling module, suppress the activation of JNK3 and the downstream signaling pathway. Thus, our results imply that over-expression of C-terminal amino acids of GluR6 induce neuroprotection against ischaemic brain injury in rat hippocampal CA1 region via suppressing proapoptosis signaling pathways, which can be an experimental foundation for gene therapy of stroke.

S-nitrosylation of PTEN Invovled in ischemic brain injury in rat hippocampal CA1 region.

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is not only a protein, but also a lipid phosphatase that can negatively regulate the serine/threonine kinase Akt. It has been reported that PTEN can be regulated by means of phosphorylation. However, whether PTEN can be regulated by another post-translational protein modification (S-nitrosylation) was not fully elucidated. In this study, we investigated the S-nitrosylation of PTEN during transient cerebral ischemia/reperfusion in rat hippocampus. Transient brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. Our data show that S-nitrosylation of PTEN was increased significantly after 12 h of reperfusion compared with sham control. Pretreatment with the inhibitor of nNOS (7-NI) and the inhibitor of iNOS could inhibit PTEN's activity and decrease S-nitrosylation of PTEN. Taken together, these results indicate that nitric oxide could regulate PTEN's activity via S-nitrosylation during transient global ischemia in rat hippocampus.

Neuroprotection of GST, an extract of traditional Chinese herb, against ischemic brain injury induced by transient brain ischemia and reperfusion in rat hippocampus.

In this study, we investigated the effect of GST, an extract of Chinese traditional herb, on transient brain ischemia/reperfusion-induced neuronal cell death. Immunoblotting was used to detect the phosphorylation of MLK, JNK and c-jun. Transient (15 minutes) brain ischemia was induced by the four-vessel occlusion in Sprague-Dawley rats. GST was administrated to the SD rats 20 minutes before ischemia or 1 hour after ischemia. Our data showed that the pretreatment of GST could inhibit phosphorylation of MLK, JNK and c-jun. Moreover, GST showed potent neuroprotective effects on ischemic brain damage in vivo and administration of it 1 hour after ischemia also achieved the protective effects. These results indicate that GST has a prominent neuroprotection action against brain ischemic damage and provides a promising therapeutic approach for ischemic brain injury.

Pergolide protects CA1 neurons from apoptosis in a gerbil model of global cerebral ischemia.

OBJECTIVE: To investigate the effects of dopamine (DA) receptor agonists and antagonists on neuronal apoptosis in hippocampal CA1 region after forebrain ischemia/reperfusion (I/R) injury in gerbils. METHODS: Gerbil forebrain ischemia was induced by occluding bilateral carotid arteries for 5 minutes. The open field test, hematoxylin-eosin staining and in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) methods were used 1, 3 and 7 days after reperfusion. Western blot was used to examine the phosphorylation of c-Jun. RESULTS: Pergolide could significantly reduce the habituation impairments of ischemic gerbils, increase the number of normal neurons and reduce the number of apoptotic neurons in hippocampal CA1 region after reperfusion. SKF38393, SCH23390 and spiperone had no effects on these changes in this transient I/R injury model. Furthermore, pergolide can significantly reduce the phosphorylation of c-Jun induced by transient forebrain ischemia.

Opposing effects of Bad phosphorylation at two distinct sites by Akt1 and JNK1/2 on ischemic brain injury.

Increasing evidence suggests that the Bcl-2 family proteins play pivotal roles in regulation of the mitochondria cell-death pathway on transient cerebral ischemia. Bad, a BH3-only proapoptotic Bcl-2 family protein, has been shown to be phosphorylated extensively on serine by kinds of kinases. However, the exact mechanisms of the upstream kinases in regulation of Bad signaling pathway remain unknown. Here, we reported that Bad could be phosphorylated not only by Akt1 but also by JNK1/2 after transient global ischemia in rat hippocampal CA1 region. Our data demonstrated that Akt1 mediated the phosphorylation of Bad at serine 136, which increased the interaction of serine 136-phosphorylated Bad with 14-3-3 proteins and prevented the dimerization of Bad with Bcl-Xl, inhibited the release of cytochrome c to the cytosol and the death effector caspase-3 activation, leading to the survival of neuron. In contrast, JNK1/2 induced the phosphorylation of Bad at a novel site of serine 128 after brain ischemia/reperfusion, which inhibited the interaction of PI3K/Akt-induced serine 136-phosphorylated Bad with 14-3-3 proteins, thereby promoted the apoptotic effect of Bad. In addition, activated Akt1 inhibited the activation of Bad(S128) through downregulating JNK1/2 activation, thus inhibiting JNK-mediated Bad apoptosis pathway. Furthermore, the fate of cell to survive or to die was determined by a balance between prosurvival and proapoptotic signals. Taken together, our studies reveal that Bad phosphorylation at two distinct sites induced by Akt1 and JNK1/2 have opposing effects on ischemic brain injury, and present the possibility of Bad as a potential therapeutic target for stroke treatment.

Crosstalk between PSD-95 and JIP1-mediated signaling modules: the mechanism of MLK3 activation in cerebral ischemia.

Our previous study indicates that global ischemia facilitates the assembly of the GluR6.PSD-95.MLK3 signaling module, which in turn activated MLK3, leading to exacerbated ischemic neuron death. In addition, JIP1, functioning as a scaffold protein, could couple MLK3-MKK7-JNK to form a specific signaling module and facilitate the activation of the JNK signal pathway. However, the organization, regulation, and function between the two signaling modules and the effects they have on MLK3 activation remain incompletely understood. Here, we show that JIP1 maintains MLK3 in an inactive and monomeric state; once activated, MLK3 binds to PSD-95 and then dimerizes and autophosphorylates. In addition, a GluR6 C-terminus-containing peptide (Tat-GluR6-9c) and antisense oligonucleotides (AS-ODNs) against PSD-95 inhibit the integration of PSD-95 and MLK3 and the dimerization of MLK3, facilitate the interaction of JIP1 and MLK3, and, consequently, perform neuroprotection on neuron death. However, AS-ODNs against JIP1 play a negative role compared to that mentioned above. The findings show that the crosstalk occurs between PSD-95 and the JIP1-mediated signaling module, which may be involved in brain ischemic injury and contribute to the regulation of MLK3 activation. Thus, specific blockade of PSD-95-MLK3 coupling may reduce the extent of ischemia-reperfusion-induced neuronal cell death.

Inhibition of proliferation and induction of apoptosis in human renal carcinoma cells by anti-telomerase small interfering RNAs.

Telomerase is an attractive molecular target for cancer therapy because it is present in most malignant cells but is undetectable in most normal somatic cells. Human telomerase consists of two subunits, an RNA component (hTR) and a human telomerase reverse transcriptase component (hTERT). Small interfering RNA (siRNA), one kind of RNA interferences, has been demonstrated to be an effective method to inhibit target gene expression in human cells. We investigated the effects of siRNA targeting at both hTR and hTERT mRNA on the inhibition of telomerase activity in human renal carcinoma cells (HRCCs). The proliferation and apoptosis of HRCCs were examined. The treatment of HRCCs using hTR and hTERT siRNAs resulted in significant decrease of hTR mRNA, hTERT mRNA and hTERT protein. The siRNA can also inhibit the telomerase activity and the proliferation of HRCCs. Moreover, they can induce apoptotic cell death in a dose-dependent manner. From these findings, we propose that the inhibition of telomerase activity using siRNA targeting hTR and hTERT might be a rational approach in renal cancer therapy.

Blockade of the translocation and activation of mitogen-activated protein kinase kinase 4 (MKK4) signaling attenuates neuronal damage during later ischemia-reperfusion.

Mitogen-activated protein kinase kinase 4 (MKK4), as an upstream activator of c-Jun NH(2)-terminal kinase (JNK), plays a critical role in response to cellular stresses and pro-inflammatory cytokines. In this study, we investigated the subcellular localization and activation of MKK4 in response to global cerebral ischemia. Our results indicated that MKK4 had two activation peaks in both the cytosol and the nucleus, and translocated from the cytosol to the nucleus at 30 min and 6 h of reperfusion. We also detected the interaction of JNK-interacting protein 3 (JIP3) and MKK4, which reached a maximum at 6 h of reperfusion. To elucidate the mechanism of translocation and activation, we administered N-acetylcysteine, an antioxidant reagent, and a glutamate receptor 6 C-terminus-containing peptide (Tat-GluR6-9c) to rats. The data showed that N-acetylcysteine limited the translocation and activation at 30 min of reperfusion; however, the peptide perturbed the subcellular localization and activation at 6 h of reperfusion, and subsequently provided a protective role against delayed neuronal cell death. Taken together, these results demonstrate that the translocation and activation of MKK4 during early reperfusion are closely associated with reactive oxygen species, whereas, at late reperfusion, MKK4 activation may be involved in brain ischemic injury.

Neuroprotection of Tat-GluR6-9c against neuronal death induced by kainate in rat hippocampus via nuclear and non-nuclear pathways.

Previous studies have suggested that glutamate receptor 6 (GluR6) subunit- and JNK-deficient mice can resist kainate-induced epileptic seizure and neuronal toxicity (Yang, D. D., Kuan, C.-Y., Whitmarsh, A. J., Rinocn, M., Zheng, T. S., Davis, R. J., Rakic, P., and Flavell, R. A. (1997) Nature 389, 865-870; Mulle, C., Seiler, A., Perez-Otano, I., Dickinson-Anson, H., Castillo, P. E., Bureau, I., Maron, C., Gage, F. H., Mann, J. R., Bettler, B., and Heinemmann, S. F. (1998) Nature 392, 601-605). In this study, we show that kainate can enhance the assembly of the GluR6-PSD95-MLK3 module and facilitate the phosphorylation of JNK in rat hippocampal CA1 and CA3/dentate gyrus (DG) subfields. More important, a peptide containing the Tat protein transduction sequence (Tat-GluR6-9c) perturbed the assembly of the GluR6-PSD95-MLK3 signaling module and suppressed the activation of MLK3, MKK7, and JNK. As a result, the inhibition of JNK activation by Tat-GluR6-9c diminished the phosphorylation of the transcription factor c-Jun and down-regulated Fas ligand expression in hippocampal CA1 and CA3/DG regions. The inhibition of JNK activation by Tat-Glur6-9c attenuated Bax translocation, the release of cytochrome c, and the activation of caspase-3 in CA1 and CA3/DG subfields. Furthermore, kainate-induced neuronal loss in hippocampal CA1 and CA3 subregions was prevented by intracerebroventricular injection of Tat-Glur6 - 9c. Taken together, our findings strongly suggest that the GluR6-PSD95-MLK3 signaling module mediates activation of the nuclear and non-nuclear pathways of JNK, which is involved in brain injury induced by kainate. Tat-GluR6-9c, the peptide we constructed, gives new insight into seizure therapy.

Treatment with vector-expressed small hairpin RNAs against Ki67 RNA-induced cell growth inhibition and apoptosis in human renal carcinoma cells.

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III promoters can trigger sequence-selective gene silencing in mammalian cells. By virtue of their excellent function in knocking down expression of cancer-associated genes, shRNAs could be used as new therapeutic agents for cancer. As overexpression of Ki67 in renal cancer has been correlated to a more aggressive tumor phenotype, inhibition of Ki67 protein expression by means of shRNAs seems to be a promising approach for the therapy of renal cancer. In this study, we constructed an expression plasmid encoding shRNAs against the Ki67 gene, named pSilencerKi67, and transfected it into human renal carcinoma cells. The pSilencerKi67 was shown to significantly knock down the expression of the Ki67 gene in human renal carcinoma cells, resulting in inhibiting proliferation and inducing apoptotic cell death that can be maintained for at least 6 d. These findings offer the promise of using vector-based shRNAs against Ki67 in renal cancer gene therapy.

Knockdown of Ki-67 by small interfering RNA leads to inhibition of proliferation and induction of apoptosis in human renal carcinoma cells.

To investigate the effect of small-interfering RNA (siRNA) targeted against Ki-67, which is an attractive molecular target for cancer therapy, on inhibiting Ki-67 expression and cell proliferation in human renal carcinoma cells (HRCCs), siRNAs were used to inhibit the expression of Ki-67 in HRCCs. Ki-67 mRNA levels were detected by RT-PCR and in situ hybridization analysis. Ki-67 protein levels were detected by Western blot and immunocytochemistry analysis. TUNEL assay was used to measure the apoptosis of carcinoma cells. Results of RT-PCR and in situ hybridization demonstrated reduction of Ki-67 mRNA expression in Ki-67 siRNAs treated 786-0 cells. Similar reduction in Ki-67 protein measured by Western blot and immunocytochemistry was observed in cells transfected with Ki-67 siRNA. Ki-67-siRNA treatment of HRCCs resulted in specific inhibition of proliferation and increased apoptotic cell death. From these findings we conclude that inhibition of Ki-67 expression by siRNA may be a reasonable approach in renal cancer therapy.

Neuroprotection against ischaemic brain injury by a GluR6-9c peptide containing the TAT protein transduction sequence.

It is well documented that N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors play a pivotal role in ischaemic brain injury. Recent studies have shown that kainate (KA) receptors are involved in neuronal cell death induced by seizure, which is mediated by the GluR6*PSD-95*MLK3 signalling module and subsequent c-Jun N-terminal kinase (JNK) activation. Here we investigate whether GluR6 mediated JNK activation is correlated with ischaemic brain injury. Our results show that cerebral ischaemia followed by reperfusion can enhance the assembly of the GluR6*PSD-95*MLK3 signalling module and JNK activation. As a result, activated JNK can not only phosphorylate the transcription factor c-Jun and up-regulate Fas L expression but can also phosphorylate 14-3-3 and promote Bax translocation to mitochondria, increase the release of cytochrome c and increase caspase-3 activation. These results indicate that GluR6 mediated JNK activation induced by ischaemia/reperfusion ultimately results in neuronal cell death via nuclear and non-nuclear pathways. Furthermore, the peptides we constructed, Tat-GluR6-9c, show a protective role against neuronal death induced by cerebral ischaemia/reperfusion through inhibiting the GluR6 mediated signal pathway. In summary, our results indicate that the KA receptor subunit GluR6 mediated JNK activation is involved in ischaemic brain injury and provides a new approach for stroke therapy.

Neuroprotective effects of GluR6 antisense oligodeoxynucleotides on transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region.

To investigate whether the kainate (KA) receptors subunit GluR6 is involved in the neuronal cell death induced by cerebral ischemia followed by reperfusion, the antisense oligodeoxynucleotides (ODNs) of GluR6 were used to suppress the expression of GluR6 by intracerebroventricular infusion once per day for 3 days before ischemia. Transient brain ischemia was induced by four-vessel occlusion in Sprague-Dawley rats. The effects of GluR6 antisense ODNs on the phosphorylation of MLK3 and JNK and the interactions of MLK3 and PSD-95 with GluR6 were examined by immunoprecipitation and immunoblotting. Our results show that GluR6 antisense ODNs can knock down the expression of GluR6 and suppress the assembly of the GluR6.PSD-95.MLK3 signaling module and, therefore, inhibit JNK activation and phosphoralation of c-jun. On the other hand, the GluR6 antisense ODNs also show a protective role against neuronal cell death induced by cerebral ischemia/reperfusion. Administration of GluR6 antisense ODNs once per day for 3 days before cerebral ischemia significantly decreased neuronal degeneration. In conclusion, our results demonstrate that kainate receptor subunit GluR6 plays an important role in neuronal death induced by cerebral ischemia followed by reperfusion.