ABSTRACT
Cancer immunotherapy has received extensive attention due to its ability to activate the innate or adaptive immune systems of patients to combat tumors. Despite a few clinical successes, further endeavors are still needed to tackle unresolved issues, including limited response rates, development of resistance, and immune-related toxicities. Accumulating evidence has pinpointed the tumor microenvironment (TME) as one of the major obstacles in cancer immunotherapy due to its detrimental impacts on tumor-infiltrating immune cells. Nanomedicine has been battling with the TME in the past several decades, and the experience obtained could be exploited to improve current paradigms of immunotherapy. Here, we discuss the metabolic features of the TME and its influence on different types of immune cells. The recent progress in nanoenabled cancer immunotherapy has been summarized with a highlight on the modulation of immune cells, tumor stroma, cytokines and enzymes to reverse the immunosuppressive TME.
Subject(s)
Immunologic Factors/pharmacology , Immunotherapy , Nanomedicine , Neoplasms/therapy , Humans , Neoplasms/immunology , Tumor Microenvironment/drug effectsABSTRACT
PURPOSE: DNA methylation plays major regulatory roles in gene transcription. Our previous studies confirmed that Ki-67 promoter is hypomethylated and Sp1 is a transcriptional activator of Ki-67 gene in cancer cells. However, whether Sp1-mediated transcriptional activation of Ki-67 is related to its methylation has not been studied yet. MATERIALS AND METHODS: In this study, we confirmed that methylated CpG binding protein 2 (MBD2) binding to methylated DNA hindered the binding of Sp1 to Ki-67 promoter and then repressed Ki-67 transcription through chromatin immunoprecipitation (ChIP) and quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation (Co-IP), ChIP, methylation-specific PCR (MS-PCR) and Western blot were utilized to analyze the effects of Sp1 binding to Ki-67 promoter on its methylation status. RESULTS: Less DNA methyltransferase 1 (DNMT1) bound to the Ki-67 promoter in MKN45 cells than in HK-2 cells. Histone acetyltransferase p300 that was recruited by Sp1 to Ki-67 promoter could attenuate the methylation level of Ki-67 promoter. Furthermore, higher expression of Sp1 and Ki-67 was related to the overall survival (OS), first progression (FP) and post-progression survival (PPS) in gastric cancer by scrutinizing bioinformatics datasets. CONCLUSION: Taken together, our findings suggested that hypomethylation of Ki-67 promoter enhanced the binding of Sp1, which in turn maintained hypomethylation of promoter, leading to increase Ki-67 expression in cancer cells. Sp1 and Ki-67 could act promising prognostic biomarkers for clinical diagnosis and treatment of cancer.
ABSTRACT
OBJECTIVES: This study aimed to investigate the safety and therapeutic efficacy of herbal drug, Di Huang Yin Zi (DHYZ), in patients affected by ischemic stroke. METHODS: In this double blind, placebo-controlled study, a total of 100 patients with recent (less than 30 days) ischemic stroke were randomized to receive DHYZ or placebo for 12 weeks. Both groups also received rehabilitation therapy during the study period. As there were 13 dropouts, a total of 45 patients on DHYZ and 42 on placebo were available for analysis. The Fugl-Meyer Assessment (FMA) and Barthel index (BI) were assessed before treatment and at 4-week intervals. RESULTS: We observed that the FMA score and BI were increased, in both groups at week 4, 8 and 12 compared with the baseline. Furthermore, significantly better FMA score was observed in patients treated with DHYZ at week 8 and 12 (both P<0.05). BI was significantly higher in DHYZ group than in placebo group at weeks 12 (P<0.05). At week 12, the 95% Confidence Intervals (CI) of mean difference of FMA and BI also indicated that the differences between two groups were statistically significant. Compared to placebo, DHYZ produced significantly greater improvement in FMA grade at week 12 (44.4% versus 23.8%, χ(2)=4.09, P<0.05). CONCLUSIONS: DHYZ showed good efficacy, safety and tolerability in patients affected by ischemic stroke. We conclude that DHYZ may be a useful therapeutic option in patients with ischemic stroke.
Subject(s)
Brain Ischemia/drug therapy , Drugs, Chinese Herbal/therapeutic use , Stroke/drug therapy , Aged , China , Female , Humans , Male , Middle Aged , PhytotherapyABSTRACT
Renal cell carcinoma is the most frequent kidney malignancy and patients with metastatic disease have a poor prognosis. Suppressed apoptosis and marked invasiveness are distinctive features of renal cell carcinoma. In the present study, a dualregulated oncolytic adenovirus expressing the interluekin (IL)24 gene (Ki67ZD55IL24) was constructed utilizing the Ki67 promoter to replace the native viral promoter of the E1A gene. Whether the combination of Ki67ZD55IL24mediated gene virotherapy and radiotherapy produced increased cytotoxicity in renal cell carcinoma cells via mitochondrial apoptotic cell death was investigated. The data indicated that this novel strategy has the potential to be further developed into an effective approach to treat renal cell carcinoma. The results showed that the combination of Ki67ZD55IL24 and radiotherapy significantly enhanced antitumour activity via increasing the induction of apoptosis in melanoma cells compared with the other agents.
Subject(s)
Adenoviridae/genetics , Apoptosis/genetics , Genetic Vectors/genetics , Mitochondria/genetics , Oncolytic Viruses/genetics , Radiation Tolerance/genetics , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/radiotherapy , Cell Line, Tumor , Cell Survival/genetics , Cell Survival/radiation effects , Humans , Interleukins/genetics , Ki-67 Antigen/genetics , Promoter Regions, GeneticABSTRACT
The expression of Ki67 is strongly associated with tumor cell proliferation and growth, and is widely used in routine pathological investigation as a proliferation marker. The nuclear protein Ki67 (pKi67) is an established prognostic and predictive indicator for the assessment of biopsies from patients with cancer. Clinically, pKi67 has been shown to correlate with metastasis and the clinical stage of tumors. In addition, it has been shown that Ki67 expression is significantly higher malignant tissues with poorly differentiated tumor cells, as compared with normal tissue. According to its predictive role, pKi67 expression identifies subpopulations of patients who are more likely to respond to a given therapy. The Ki67 labeling index is an independent prognostic factor for survival rate, which includes all stages and grade categories. There is a correlation between the ratio of Ki67positive malignant cells and patient survival. It has been shown that blocking of Ki67 either by microinjection of antibodies or through the use of antisense oligonucleotides leads to the arrest of cell proliferation. Specifically, antisense oligonucleotides and antibodies against pKi67 have been shown to inhibit the progression of the cell cycle. The Ki67 protein is well characterized at the molecular level and is extensively used as a prognostic and predictive marker for cancer diagnosis and treatment. Increasing evidence indicates that Ki67 may be an effective target in cancer therapy. It therefore merits further development, including testing in more sophisticated in vitro and appropriate in vivo models. This review provides an overview of recent advances in this field.
Subject(s)
Biomarkers, Tumor , Ki-67 Antigen/metabolism , Neoplasms/diagnosis , Neoplasms/metabolism , Animals , Humans , Ki-67 Antigen/genetics , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/genetics , PrognosisABSTRACT
Melanoma is the most malignant skin cancer and is highly resistant to chemotherapy and radiotherapy. Curcumin is a component of turmeric, the yellow spice derived from the rhizome of Curcuma longa. It has been demonstrated to modulate multiple cell signaling pathways, including apoptosis, proliferation, angiogenesis and inflammation. In this study, we studied the signaling pathways involved in melanoma cell death after treatment with curcumin using western blotting. Colorimetric assays (MTT) assessed cell viability. Flow cytometry and DNA laddering evaluated cell apoptosis. Fluorescent microscopy was used to evaluate of Hoechst 33342 staining of nuclei. The result demonstrated that curcumin could induce apoptosis and inhibit proliferation in melanoma cells. Curcumin stimulated the expression of pro-apoptotic Bax, and inhibited the activation of anti-apoptotic Mcl-1 and Bcl-2. During curcumin treatment, caspase-8 and Caspase-3 were cleaved in time and dose-dependent manners. Curcumin treatment also altered the expressions of apoptosis associated proteins NF-κB, p38 and p53. Curcumin induced DNA double strand breaks, which were indicated by phosphorylated H2AX. Our data suggested that curcumin could be used as a novel and effective approach for the treatment of melanoma.
Subject(s)
Apoptosis/drug effects , Caspases/metabolism , Curcumin/pharmacology , Melanoma/enzymology , Melanoma/pathology , Mitochondria/metabolism , Annexin A5/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Curcumin/chemistry , DNA Breaks, Double-Stranded/drug effects , DNA Fragmentation/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Flow Cytometry , Humans , Mitochondria/drug effects , NF-kappa B/metabolism , Propidium/metabolism , Time Factors , Tumor Suppressor Protein p53/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) induces caspase-3 cleavage and subsequent activation via the intrinsic or extrinsic pathway to result in cancer cell-selective apoptosis, but whether mda-7/IL-24 may directly regulate caspase-3 through the post-translational modification remains unknown. Here, we reported that tumor-selective replicating adenovirus ZD55-IL-24 led to caspase-3 denitrosylation and subsequent activation, indicating that caspase-3 denitrosylation played a crucial role in ZD55-IL-24-induced cancer cell apoptosis. To confirm the relationship between caspase-3 denitrosylation and its activation in response to ZD55-IL-24, we treated carcinoma cells with the different nitric oxide (NO) regulators to modulate caspase-3 denitrosylation level, then observed the corresponding caspase-3 cleavage. We found that NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) promoted caspase-3 denitrosylation and caspase-3 cleavage, thereby exacerbating ZD55-IL-24-induced cancer cell apoptosis, whereas NO donor sodium nitroprusside (SNP) showed the opposite effect. Moreover, caspase-3 denitrosylation facilitated its downstream target poly ADP-ribose polymerase (PARP) degradation that further increased the apoptotic susceptibility. Although caspase-3 activation controlled by denitrosylation modification has emerged as an important regulator of programmed cell death, the detailed molecular mechanism by which caspase-3 exerts its denitrosylation modification in response to ZD55-IL-24 still needs to be elucidated. Thus, our results demonstrated that ZD55-IL-24 increased Fas expression to enhance thioredoxin reductase 2 (TrxR2), which was responsible for caspase-3 denitrosylation. Collectively, these findings elucidate that ZD55-IL-24 induces caspase-3 denitrosylation through Fas-mediated TrxR2 enhancement, thereby facilitating caspase-3 cleavage and the downstream caspase signaling pathway activation, which provides a novel insight into ZD55-IL-24-induced cancer-specific apoptosis by post-translational modification of the apoptotic executor caspase-3.
Subject(s)
Caspase 3/metabolism , Interleukins/metabolism , Adenoviridae/genetics , Apoptosis , Cloning, Molecular , Gene Transfer Techniques , HeLa Cells , Humans , Interleukins/genetics , Organ Specificity , Protein Processing, Post-Translational/genetics , Signal Transduction/genetics , Thioredoxin Reductase 2/metabolism , Transgenes/genetics , fas Receptor/metabolismABSTRACT
Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O(6)-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Dacarbazine/analogs & derivatives , Drug Resistance, Neoplasm/genetics , O(6)-Methylguanine-DNA Methyltransferase/genetics , Research , Animals , Antineoplastic Agents, Alkylating/therapeutic use , DNA Methylation , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Gene Expression , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/drug therapy , Neoplasms/genetics , O(6)-Methylguanine-DNA Methyltransferase/antagonists & inhibitors , Promoter Regions, Genetic , TemozolomideABSTRACT
The aim of our study was to elucidate the role of Rap2B in the development of human suprarenal epithelioma and to investigate the effect of Rap2B on suprarenal epithelioma cells migration and invasion. We use tissue microarray and immunohistochemistry to evaluate Rap2B staining in 75 suprarenal epithelioma tissues and 75 tumor-adjacent normal renal tissues. And the expression of Rap2B protein in human suprarenal epithelioma cells and tissues was detected by western blot simultaneously. The role of Rap2B in suprarenal epithelioma cells migration and invasion was detected by using wound healing assay, cell migration assay, and matrigel invasion assay. After that, we performed western blot analysis and gelatin zymography to detect MMP-2 protein expression and enzyme activity. Our research showed that Rap2B expression was increased in tumor tissues compared with tumor-adjacent normal renal tissues. But no correlation was found between Rap2B expression and clinicopathological parameters. In addition, we found that Rap2B promoted the cell migration and invasion abilities, and Rap2B increased MMP-2 expression and enzyme activity in suprarenal epithelioma cells. Our data indicated that Rap2B expression is significantly increased in human suprarenal epithelioma and Rap2B can promote the cell migration and invasion abilities, which may provide a new target for the treatment of suprarenal epithelioma.
Subject(s)
Carcinoma, Renal Cell/metabolism , Cell Movement , Kidney Neoplasms/metabolism , rap GTP-Binding Proteins/metabolism , Blotting, Western , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Female , Humans , Immunohistochemistry , Kidney Neoplasms/genetics , Kidney Neoplasms/pathology , Male , Matrix Metalloproteinase 2/metabolism , Middle Aged , Neoplasm Invasiveness , RNA Interference , Tissue Array Analysis , rap GTP-Binding Proteins/geneticsABSTRACT
Oncolytic adenoviruses are a novel class of anticancer treatment, based upon their ability to replicate selectively within malignant cells resulting in cell lysis. The replicationselective adenovirus, ZD55IL24, was constructed by harboring an E1B55 kDa deletion and arming with interleukin-24 (IL-24). The microtubulestabilizing drug paclitaxel (PTX) exhibits activity in relapsed cancer. In the present study, the synergistic antitumor effects of the combination of PTX and ZD55IL24 on breast cancer cells was investigated. The results demonstrated that there were different roles for PTX in the expression of transgenic mRNA and protein. ZD55IL24 combined with PTX induced marked growth inhibition of MDAMB231 and Bcap37 cells. PTX increased viral uptake and appeared not to alter the replication of ZD55IL24 in breast cancer cells. Annexin Vfluorescein isothiocyanate/propidium iodide staining and the Hoechst 33258 assay indicated that ZD55IL24 induced an increase in the number of apoptotic cells when administered in combination with PTX. It was demonstrated that ZD55IL24 conjugated with PTX was highly concomitant, and increased proapoptotic proteins levels, activated caspase3, -7 and -9 and downregulated antiapoptotic proteins. These results suggested that ZD55IL24 in combination with PTX exhibited a markedly increased cytotoxic and apoptosisinducing effect in breast cancer cells. Thus, this chemogeneviro therapeutic strategy was demonstrated to be superior to conventional chemotherapy or geneviro therapy alone.
Subject(s)
Adenoviridae/genetics , Antineoplastic Agents, Phytogenic/therapeutic use , Breast Neoplasms/therapy , Interleukins/genetics , Oncolytic Virotherapy , Paclitaxel/therapeutic use , Apoptosis , Blotting, Western , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation , Combined Modality Therapy , Drug Synergism , Female , Humans , Prohibitins , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Virus Replication/drug effectsABSTRACT
Malignant melanoma is one of the most lethal and aggressive human malignancies. Suppressed apoptosis and extraordinary invasiveness are the distinctive features that contribute to malignant melanoma. The alkylating agent temozolomide (TMZ) is one of the most effective single chemotherapeutic agents for patients with malignant melanoma, but resistance develops quickly and with high frequency. We constructed a dual-regulated oncolytic adenovirus expressing interleukin 24 (IL-24) gene (Ki67-ZD55-IL-24) by utilizing the Ki67 promoter to replace the native viral promoter of E1A gene. We investigated whether a combination of Ki67-ZD55-IL-24-mediated gene virotherapy and chemotherapy using TMZ produces increased cytotoxicity against human melanoma cells via the induction of apoptosis. Our data indicate that this novel strategy thus holds promising potentials for further developing an effective approach to treat malignant melanoma.
Subject(s)
Adenoviridae/genetics , Antineoplastic Agents, Alkylating/pharmacology , Apoptosis , Dacarbazine/analogs & derivatives , Interleukins/genetics , Melanoma/therapy , Oncolytic Virotherapy , Blotting, Western , Cell Cycle , Cell Proliferation , Combined Modality Therapy , DNA Modification Methylases/metabolism , DNA Repair Enzymes/metabolism , Dacarbazine/pharmacology , Genetic Therapy , Genetic Vectors , Humans , Immunoenzyme Techniques , Ki-67 Antigen/genetics , Melanoma/genetics , Melanoma/pathology , Melanoma/virology , Temozolomide , Tumor Cells, Cultured , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/metabolismABSTRACT
Adiponectin secreted from adipose tissues plays a role in the regulation of energy homeostasis, food intake, and reproduction in the hypothalamus. We have previously demonstrated that adiponectin significantly inhibited GNRH secretion from GT1-7 hypothalamic GNRH neuron cells. In this study, we further investigated the effect of adiponectin on hypothalamic KISS1 gene transcription, which is the upstream signal of GNRH. We found that globular adiponectin (gAd) or AICAR, an artificial AMPK activator, decreased KISS1 mRNA transcription and promoter activity. Conversely, inhibition of AMPK by Compound C or AMPKα1-SiRNA augmented KISS1 mRNA transcription and promoter activity. Additionally, gAd and AICAR decreased the translocation of specificity protein-1 (SP1) from cytoplasm to nucleus; however, Compound C and AMPKα1-siRNA played an inverse role. Our experiments in vivo demonstrated that the expression of Kiss1 mRNA was stimulated twofold in the Compound C-treated rats and decreased about 60-70% in gAd- or AICAR-treated rats compared with control group. The numbers of kisspeptin immunopositive neurons in the arcuate nucleus region of Sprague Dawley rats mimicked the same trend seen in Kiss1 mRNA levels in animal groups with different treatments. In conclusion, our results provide the first evidence that adiponectin reduces Kiss1 gene transcription in GT1-7 cells through activation of AMPK and subsequently decreased translocation of SP1.
Subject(s)
Adenylate Kinase/physiology , Adiponectin/pharmacology , Hypothalamus/drug effects , Kisspeptins/genetics , Neurons/drug effects , Sp1 Transcription Factor/physiology , Adenylate Kinase/metabolism , Adiponectin/physiology , Aminoimidazole Carboxamide/analogs & derivatives , Aminoimidazole Carboxamide/pharmacology , Animals , Cells, Cultured , Down-Regulation/drug effects , Down-Regulation/genetics , Female , Hypothalamus/cytology , Hypothalamus/metabolism , Kisspeptins/metabolism , Neurons/metabolism , Phosphorylation/drug effects , Protein Transport/drug effects , Protein Transport/genetics , Protein Transport/physiology , Rats , Rats, Sprague-Dawley , Ribonucleotides/pharmacology , Sp1 Transcription Factor/metabolism , Transcription, Genetic/drug effectsABSTRACT
Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has shown potent tumor cell apoptosis inducing capacity in multiple cancers. However, the apoptosis induction capacity of mda-7/IL-24 was low and directly correlated with the adhesion to tumor cells.Cell adhesion molecule integrin α(v)ß(3) expressed on the surface of several types of solid tumor cells, and they bind to arginine-glycine-aspartic acid (RGD) which enhanced the adhesion to tumor cells. This rout was exploited to construct a tumor-targeting gene RGD-IL-24 which can express RGD-MDA-7/IL-24 protein that includes the cell adhesive sequence (164)Arg-(165)Gly-(166)Asp (A Glycine residue was inserted into the recombinant MDA-7/IL-24 between Arg164 and Asp165 to form a RGD motif). We successfully got the MDA-7/IL-24 mutant by overlapping polymerase chain reaction (PCR) and evaluated its therapeutic efficacy for tumor cell lines MCF-7, HeLa, HepG2, and normal human lung fibroblast (NHLF) line. And we found that the expression of pCDNA3.1/RGD-IL-24 was same to the expression of pCDNA3.1/IL-24. The RGD-IL-24 enhanced the apoptosis-inducing function in tumor cells, but not in normal cells. In tumor cell lines, the apoptosis-inducing activities of RGD-IL-24 was significantly higher than IL-24 detecting by MTT assay, Annexin V, and Hoechst 33258 analysis. Further, pCDNA3.1/RGD-IL-24 showed a significant increase in the ratio of pro-apoptotic (bax) to anti-apoptotic (bcl-2) proteins in tumor cell lines, but not in NHLF cell line. Together, these results suggest that RGD-IL-24 can enhance the apoptosis of tumor cells and may provide a promising drug in tumor therapy.
Subject(s)
Apoptosis , Gene Expression Regulation, Neoplastic , Interleukins/biosynthesis , Mutation , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Oligopeptides , Cell Adhesion/genetics , Cell Line, Tumor , HeLa Cells , Hep G2 Cells , Humans , Integrin alpha5/biosynthesis , Integrin alpha5/genetics , Integrin beta3/biosynthesis , Integrin beta3/genetics , Interleukins/genetics , Neoplasm Proteins/genetics , Neoplasms/genetics , Neoplasms/pathology , Neoplasms/therapy , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
The expression of the human Ki-67 protein, which is strictly associated with cell proliferation, is regulated by a variety of cellular mediators. In this study, we studied the effects of p53 on Ki-67 promoter in HeLa cells using luciferase reporter assay. The results showed that: (1) p53 inhibited Ki-67 promoter activity in a dose-dependent manner, (2) the p53-binding motifs mediated part of the transcriptional repression of Ki-67 promoter through a sequence-specific interaction with p53, (3) p53 was able to repress the Sp1-stimulated Ki-67 promoter activity, and (4) the Sp1-binding sites were responsible for the p53-mediated transcriptional repression of Ki-67 promoter. In conclusion, p53 inhibited Ki-67 promoter activity via p53- and Sp1-dependent pathways, and the interaction between p53 and Sp1 might be involved in the transcriptional regulatory mechanisms.
Subject(s)
Gene Expression Regulation/physiology , Ki-67 Antigen/biosynthesis , Sp1 Transcription Factor/metabolism , Tumor Suppressor Protein p53/metabolism , Down-Regulation , Gene Expression , HeLa Cells , Humans , Immunohistochemistry , Ki-67 Antigen/genetics , Promoter Regions, Genetic , Reverse Transcriptase Polymerase Chain Reaction , Sp1 Transcription Factor/genetics , Transfection , Tumor Suppressor Protein p53/geneticsABSTRACT
To investigate the effect of methylated oligonucleotide (MON) targeting Ki-67 promoter on the expression of Ki-67 gene and the proliferation and apoptosis of the human 786-0 renal carcinoma cells, human 786-0 cells were transfected with MON. The activity of Ki-67 promoter was detected by dual-luciferase reporter assay system. Among the five methylated oligonucleotides (MON(1)-MON(5)), MON(4) is the best excellent one in the inhibition of the Ki-67 promoter activity. The activity of Ki-67 promoter is decreased to 77.88% in 40-nM group, 50.07% in 80-nM group, 35.63% in 120-nM group, 26.09% in 160-nM group, and 16.98% in 200-nM group compared with 0-nM group. The activity of Ki-67 promoter in MON group is decreased to 61.96% at 8 h, 48.93% at 12 h, 15.97% at 24 h, 26.00% at 36 h, 35.01% at 48 h, 46.08% at 72 h, and 66.12% at 96 h compared with pGLBK235 group. These results show that the effect of MON is time- and dose-dependent. The activity of Ki-67 in MON group is decreased to 16.73% compared with pGLBK235 group, while the control groups have no significant difference. The expression of Ki-67 gene in 786-0 cells was detected by RT-PCR and immunohistochemistry, respectively. The expression of Ki-67 mRNA is decreased to 61.04% and that of Ki-67 protein is decreased to 32.07% in MON group compared with the blank group. The proliferation of 786-0 cells was determined by WST-8. The cell proliferation in MON group is decreased to 61.02% at 24 h, 73.78% at 48 h, 79.72% at 72 h, and 91.53% at 96 h compared with the blank group. The cell apoptosis was measured by annexin V and propidium iodide. The number of apoptosis cells in MON group is 2.42 times of that in the blank group at earlier period and 2.57 times at mid-anaphase. We detected the effect of MON on the expression of bax and p53 by Western blot. Compared with the blank group, the expression of bax protein in MON group is increased by 66.12%, while the expression of p53 is decreased to 67.31%. Our study demonstrates that the methylated oligonucleotide targeting Ki-67 promoter has a remarkable effect on the inhibition of Ki-67 expression and the proliferation of the human 786-0 renal carcinoma cells and can induce apoptosis of the 786-0 cells.
Subject(s)
Carcinoma, Renal Cell/genetics , DNA Methylation/genetics , Gene Expression/drug effects , Ki-67 Antigen/genetics , Kidney Neoplasms/genetics , Oligonucleotides/pharmacology , Apoptosis/drug effects , Apoptosis/genetics , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , CpG Islands/genetics , Gene Expression/genetics , Humans , Immunohistochemistry , Ki-67 Antigen/metabolism , Oligonucleotides/genetics , Promoter Regions, Genetic/genetics , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , TransfectionABSTRACT
OBJECTIVE: The aim of the study was to evaluate whether or not there exists nociceptive and non-nociceptive hypersensitivity at latent myofascial trigger points (MTrPs). METHODS: Eleven healthy volunteers participated in this study, which consisted of 3 sessions of electromyography-guided intramuscular injection with a minimum of a week interval in between. In each session, a bolus of either hypertonic saline (6%, 0.1 mL, each), glutamate (0.1 mL, 0.5 M, each), or isotonic saline (0.9%, 0.1 mL, each) was randomly injected into a latent MTrP and a non-MTrP located in the right or left gastrocnemius medialis muscles. After each injection, participants were asked to rate the perceived pain intensity on an electronic visual analog scale (VAS) and to mark the pain areas on pain drawings. Maximal pain intensity (VAS(peak)), the area under the curve (VAS(auc)), and local and referred pain areas were extracted. RESULTS: Injections of either hypertonic saline, glutamate, or isotonic saline into the latent MTrPs induced a higher VAS(peak) and larger VAS(auc) than the non-MTrPs (all, P<0.05). Furthermore, the MTrPs with referred pain after painful injections were found to show higher VAS(peak) and larger VAS(auc) than those without referred pain (both, P<0.001). CONCLUSIONS: These results confirm the existence of nociceptive hypersensitivity at latent MTrPs and provide the first evidence that there exists non-nociceptive hypersensitivity (allodynia) at latent MTrPs. Finally, the occurrence of referred muscle pain is associated with higher pain sensitivity at latent MTrPs.
Subject(s)
Hyperalgesia/complications , Myofascial Pain Syndromes/complications , Pain Threshold/physiology , Adult , Chi-Square Distribution , Electromyography/methods , Female , Glutamic Acid/adverse effects , Humans , Injections, Intramuscular , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiopathology , Myofascial Pain Syndromes/chemically induced , Pain Measurement/methods , Pain Threshold/drug effects , Pain, Referred/complications , Saline Solution, Hypertonic/adverse effects , Young AdultABSTRACT
OBJECTIVES: Depression occurs frequently in post-stroke patients and appears to be associated with impairment of their rehabilitation and functional recovery. In this study, we evaluated the efficacy and tolerability of the herbal drug, Free and Easy Wanderer Plus (FEWP), in patients affected by post-stroke depression (PSD). METHODS: One hundred fifty (150) moderately to severely depressed patients as determined by a score >20 on the Hamilton Depression Scale (HDS) after a single ischemic or hemorrhagic stroke were randomly divided into the FEWP group (n = 60), the fluoxetine group (n = 60), and the placebo group (n = 30). The FEWP, fluoxetine, and placebo were administered to the patients over a period of 8 weeks. Depression was evaluated by HDS and the Barthel Index (BI) before, during, and after the treatment. RESULTS: Significantly higher clinical response rates were observed in both the FEWP and fluoxetine groups compared to the placebo group (60% and 65.5% versus 21.4%, chi(2) = 15.9, p < 0.01) and there was no difference in the response rates between the FEWP group and the fluoxetine group at the end of this study (60% versus 65.5%, chi(2) = 0.38, p > 0.05). Compared to fluoxetine, FEWP produced significantly greater improvement in depression at week 2 (15% versus 3.3%, chi(2) = 4.9, p < 0.05). Furthermore, FEWP produced significantly greater improvement in the activities of daily living (ADL) than fluoxetine at the end of this trial (BI: 43.8 +/- 5.6 versus 40.7 +/- 3.7, p < 0.01). CONCLUSIONS: FEWP showed good efficacy, safety, and tolerability in PSD patients. We conclude that FEWP is well tolerated and may be a useful therapeutic option in patients with PSD.
Subject(s)
Antidepressive Agents/administration & dosage , Depressive Disorder/drug therapy , Drugs, Chinese Herbal/administration & dosage , Phytotherapy/methods , Stroke/complications , Activities of Daily Living , Aged , Antidepressive Agents/adverse effects , China , Depressive Disorder/etiology , Double-Blind Method , Drug Therapy, Combination , Drugs, Chinese Herbal/adverse effects , Female , Fluoxetine/administration & dosage , Follow-Up Studies , Humans , Male , Middle Aged , Phytotherapy/adverse effects , Recovery of Function/drug effects , Research Design , Stroke/drug therapy , Stroke Rehabilitation , Time Factors , Treatment OutcomeABSTRACT
OBJECTIVE: To study the protective mechanism of captopril in diabetic cardiomyopathy by means of DNA microarray. METHODS: Rat models of diabetic cardiomyopathy were divided into test and control groups (n=5), and the rats in the test group were given oral captopril (1.5 mg/kg b.w.) for 15 weeks. DNA microarray was prepared by blotting the PCR products of 4 000 rat cDNAs onto a specially treated glass slides. The probes were prepared by labeling the mRNA from the myocardial tissue of both control and test groups with Cy3-d UTP and Cy5-d UTP separately through reverse transcription. The arrays were then hybridized against the cDNA probes and the fluorescent signals scanned. RESULTS: The expression of genes in relation to fatty acid b oxidation, mitochondrial proton-electron coupling and oxidative phosphorylation, and that of dithiolethione-inducible gene-1 were up-regulated, while the dimethylarginine dimethylaminohydrolase gene expression was obviously lowered in the test group in comparison with those of the control group. CONCLUSION: Captopril may protect the myocardial tissue through improving myocardial energy supply and depressing inflammatory reaction.