Heterogeneity and prognosis of programmed cell death-ligand 1 expression in the circulating tumor cells of non-small cell lung cancer.

Due to tumor heterogeneity, the consistency of programmed cell death-ligand 1 (PD-L1) expression between circulating tumor cells (CTCs) and tissue is controversial. This study aimed to establish a method for detecting CTC PD-L1 expression and exploring the impact of the same on the prognosis of lung cancer. In 32 patients with non-small cell lung cancer, lung cancer cells in the blood were enriched using CD326 immunomagnetic beads. Goat anti-mouse polyclonal CD326 antibody stained the epithelial lung cancer cells and anti-PD-L1 antibody was used to detect the expression of CTC PD-L1. The DAKO Link 48 automatic staining device detected the expression in lung cancer tissue. The consistency of PD-L1 expression was analyzed in lung cancer tissue and CTCs. The effect of plasma interferon gamma, tumor necrosis factor alpha, and interleukin-2 on PD-L1 expression and prognosis was analyzed. The number of CTCs detected in patients was 1-36, with a median of 2. There was no significant difference in PD-L1 expression fractions between CTCs and paired tumor tissue (p>0.05). The correlation coefficient was 0.20. Regardless of lung cancer tissue or CTCs, there was no statistically significant difference in the blood cytokine levels between the two groups with positive or negative PD-L1 expression (p>0.05). There was no correlation between CTCs and PD-L1 in 23 untreated patients. The expression of PD-L1 in CTCs and lung cancer tissue is heterogeneous and unaffected by the peripheral cytokines' levels. PD-L1 expression has no correlation between CTCs and tissues and is not related to prognosis.

[Overexpression of miR-382-5p Mediated Downregulation of PTEN on Malignant Biological Behavior of Human Glioma U251 Cells].

OBJECTIVE: To investigate the effect of overexpression of miR-382-5p overexpression on malignant biological behavior of human glioma U251 cells. METHODS: U251 cells were transfected with miR-382-5pmimic. Then miR-382-5p and PTEN mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) after transfection. Used bioinformatics to predicted the presence of base binding sites between miR-382-5p and PTEN, and constructed PTEN pcDNA vector overexpression plasmid was constructed. Luciluciase reporting experiment was used to detect the targeting relationship between miR-382-5p and PTEN. Cells were randomly divided into four groups: control group, mimics group, pc-PTEN group and mimics+pc-PTEN group for follow-up experiments. RT-PCR was carried out to detect the level of PTEN mRNA in each group. Cell proliferation was detected by clone formation method. The mRNA levels of Ki67, Survivin and c-Myc were detected by RT-PCR. Transwell experiment was used to assayed cell invasion ability. The expression levels of E-cadherin, N-cadherin and Vimentin were determined by Western blot. RESULTS: Results showed that miR-382-5p directly targeted PTEN. Compared with the control group, miR-382-5p and c-Myc mRNA levels and E-cadherin protein level were increased (P<0.05),PTEN, Ki67 and Survivin mRNA levels were decreased (P<0.05), cell clonal formation rate and cell invasion number were decreased (P<0.05), N-cadherin and Vimentin protein levels were decreased (P<0.05) in the mimics group; In pc-PTEN group, miR-382-5p mRNA and c-Myc mRNA levels and E-cadherin protein level were decreased (P<0.05),PTEN, Ki67 and Survivin mRNA levels were increased (P<0.05), cell clonal formation rate and cell invasion number were increased (P<0.05), N-cadherin and Vimentin protein levels were increased (P<0.05). Compared with pc-PTEN group, PTEN, Ki67 and Survivin mRNA levels, the cell clone formation rate, the number of invasion cells and the N-cadherin and Vimentin levels of mimics+PC-PTEN group decreased significantly (P<0.05), while the c-Myc mRNA level and E-cadherin protein level increased significantly (P<0.05). CONCLUSION: Overexpression of miR-382-5p mediates the downregulation of PTEN expression, causing the inhibition of the proliferation, invasion, growth and EMT of U251 glioma cells.

CBX3 promotes glioma U87 cell proliferation and predicts an unfavorable prognosis.

BACKGROUND: Chromobox protein homolog 3 (CBX3) is one of the heterochromatin protein 1 (HP1) family members. Among multiple cancers, it is usually overexpressed. However, the mechanism and function of CBX3 in glioma remain incompletely illustrated. METHODS: The expression level of CBX3 as well as its correlation with glioma are detected through TCGA and Oncomine database. The expressions of CBX3 mRNA and protein in glioma tissues and normal brain tissues have been identified by qRT-PCR and Western blot. The prognostic role of CBX3 has been assessed in a retrospective cohort study. Additionally, the correlation between CBX3 expression and the clinicopathological characteristics of glioma patients were also discussed. To better understand the role of CBX3 in glioma, a lentiviral vector expressing CBX3-shRNA and cyclin dependent kinase inhibitor 1A (CDKN1A) siRNA were established and transfected into the glioma U87 cells. Besides, the CBX3 and CDKN1A expression levels in glioma U87 cells after transfected with CBX3-shRNA were gauged by qRT-PCR and Western blot. CCK-8, colony formation and EdU assays have been applied to evaluate the influence of CBX3 on U87 cells proliferation, and flow cytometry has been applied to manage the changes in cell cycle and cell apoptosis after transfection with CBX3-shRNA. Xenograft tumors have been examined in vivo for the carcinogenic effects and prognostic value of CBX3 in glioma tissues. RESULTS: In the present study, CBX3 was demonstrated to be highly expressed in human glioma tissues, and high CBX3 expression predicted the dismal recurrence-free survival (RFS) and poor overall survival (OS) for glioma patients. High CBX3 expression was dependent on the tumor size, Karnofsky performance scale (KPS) score, WHO grade, recurrence and survival status. Moreover, CBX3 expression knockdown could remarkably suppress the proliferation and colony formation ability of U87 cells, which was achieved through blocking cell arrest at G0/G1 phase and inducing apoptosis. Additionally, our findings also suggested that, compared with shRNA-Ctrl transfection, the mRNA and protein expression levels of CDKN1A have been dramatically up-regulated in vitro after transfection with shRNA-CBX3. Consistent with the results of in vitro assays, the outcomes of xenograft assay and immunohistochemistry (IHC) also indicated that, the tumor growth and Ki-67 expression level were restrained in response to CBX3 inhibition, while the CDKN1A expression level in vivo was up-regulated. Down-regulation of CDKN1A expression partially restored the ability of cell proliferation in the U87 cells, which was inhibited by shRNA-CBX3 CONCLUSIONS: In conclusion, results of the current research suggest that a high CBX3 expression level predicts the poor prognosis for glioma patients. CBX3 can stimulate the growth of glioma U87 cells through targeting CDKN1A and CBX3 may become a novel target in the clinical treatment for glioma.

[Expression and clinical significance of ING4 and HIF-1 alpha in brain astrocytoma].

OBJECTIVE: To study the mRNA expression level of growth inhibition factor 4 (ING4) and hypoxia inducing factor 1 alpha (HIF-1 alpha), and their relationship with tumor malignant degree or the pathology classification in human brain astrocytoma. METHODS: The mRNA expression levels of ING4 and HIF-1 alpha were detected by RT-PCR method in 45 cases of grade I-IV human brain astrocytoma and 11 cases of control brain tissues from January 2009 to June 2010 in the Second Affiliated Hospital of Zhengzhou University, and their correlation was also analyzed. RESULTS: In the non-tumor brain tissue, the expression level of ING4 mRNA was 1.19 ± 0.22, while they were 0.91 ± 0.19, 0.74 ± 0.28, 0.54 ± 0.33 and 0.22 ± 0.19 in I-IV grade astrocytoma, respectively. Compared with the non-tumor control, the mRNA expression level of ING4 gene decreased significantly in the astrocytoma (P<0.05). And the expression of ING4 gradually reduced with the increase of the pathological classification of the astrocytoma.In the non-tumor brain tissue, the expression level of HIF-1 alpha mRNA was 0.26 ± 0.16, and they were 0.34 ± 0.19, 0.50 ± 0.23, 0.96 ± 0.15 and 1.04 ± 0.15 in I-IV grade astrocytoma, respectively.For HIF-1 alpha gene, the mRNA expression level increased significantly in the astrocytoma. Meanwhile, the expression gradually increased with the increase of the pathological classification of the astrocytoma (P<0.05). The mRNA expression showed a negative correlation between ING4 and HIF-1 alpha with the increase of the tumor malignant degree. CONCLUSION: The ING4 and HIF-1 alpha genes play a role in the tumorigenesis and development of human brain astrocytoma, and closely associate with the malignant degree of astrocytoma.

Study on chemotherapeutic sensitizing effect of nimotuzumab on different human esophageal squamous carcinoma cells.

Esophageal cancer is one of the leading causes of mortality worldwide. Although, surgery, radio- and chemotherapy are used to treat the disease, the identification of new drugs is crucial to increase the curative effect. The aim of the present study was to examine the chemotherapeutic sensitizing effect of nimotuzumab (h-R3) and cisplatin cytotoxic drugs cisplatin (DDP) and 5-fluorouracil (5-FU) on esophageal carcinoma cells with two different epidermal growth factor receptor (EGFR) expressions. The expression of EGFR was detected in the human EC1 or EC9706 esophageal squamous cell carcinoma cell line using immunohistochemistry. The inhibitory effect of DDP and 5-FU alone or combined with h-R3 on EC1 or EC9706 cell proliferation was detected using an MTT assay. Flow cytometry and the TUNEL assay were used to determine the effect of single or combined drug treatment on cell apoptosis. The results showed that the expression of EGFR was low in EC1 cells but high in EC9706 cells. The inhibitory effect of the single use of h-R3 on EC1 or EC9706 cell proliferation was decreased. The inhibitory effect between single use of h-R3 alone and combined use of the chemotherapy drugs showed no statistically significant difference (P>0.05) on the EC1 cell growth rate, but showed a statistically significant difference (a=0.05) on EC9706 cell growth rate. The results detected by flow cytometry and TUNEL assay showed that the difference between single use of h-R3 alone and the control group was statistically significant with regard to the EC1 apoptosis rate effect (P<0.05), but not statistically significant for EC9706 (P>0.05). However, statistically significant differences were identified in the apoptotic rate of EC9706 cells between the h-R3 combined chemotherapy group and single chemotherapy group (P<0.05), but not on in the EC1 chemotherapy group (P>0.05). In conclusion, the sensitization effect of h-R3 on chemotherapy drugs is associated with the expression level of EGFR in EC1 or EC9706 cells. The cell killing effect of the combined use of h-R3 with DDP and 5-FU showed no obvious synergistic effect compared to the single-drug group, but only an additive effect.

Inhibitory effect of DNA topoisomerase inhibitor isoliquiritigenin on the growth of glioma cells.

OBJECTIVE: To investigate the effect of isoliquiritigenin on the activity of DNA topoisomerase (TOP I) and its inhibitory effect on the growth of U87 glioma cells. METHODS: This study investigated the inhibitory effect of isoliquiritigenin on the growth of U87 glioma cells and its cytotoxicity by MTT method and determined the effect of isoliquiritigenin on TOP I activity by agarose gel electrophoresis. On this basis, we studied the interaction between isoliquiritigenin and TOP I and DNA. Finally, we further discussed the effect of isoliquiritigenin on the activity of Caspase 3, the apoptosis protein of U87 glioma cells. RESULTS: Isoliquiritigenin could inhibit the growth of U87 glioma cells (half inhibitory concentration IC50: 0.221 mM) and is of low cytotoxicity to normal cells. Agarose gel electrophoresis showed that isoliquiritigenin had significant inhibitory effect on TOP I activity. Molecular simulation results indicated that isoliquiritigenin took priority of binding to the active center of TOP I, and formed hydrogen bonds with the catalytic site Try723. Finally, Caspase 3 activity detection results suggested that isoliquiritigenin could significantly increase the activity of Caspase 3 (P < 0.05). CONCLUSION: Isoliquiritigenin had a reversible inhibitory effect on TOP I activity, reduced the rate of single strand DNA unwinding in tumor cells, and thus played an important role in inducing the apoptosis of U87 glioma cells.

Preparation of novel (-)-gossypol nanoparticles and the effect on growth inhibition in human prostate cancer PC-3 cells in vitro.

The aim of the present study was to investigate the antitumor effects and possible mechanism of (-)-gossypol nanoparticles, loaded with vv polyethylene glycol-maleimide (mPEG-Mal), in vitro. Emulsification-volatilization was used to prepare the loaded (-)-gossypol nanoparticles. The toxicity of blank nanoparticles on human prostate cancer PC-3 cells and human prostate RWPE-1 cells was measured. The antitumor effects of the nanoparticles on PC-3 cells were evaluated by an MTT assay, acridine orange staining and transmission electron microscopy in vitro, and the results were compared with those of free (-)-gossypol. In addition, the mRNA expression levels of Bcl-2 and Bak were measured using semi-quantitative reverse transcription polymerase chain reaction. The growth inhibition activity of the loaded (-)-gossypol nanoparticles was found to be dose- and time-dependent, and similar to the activity of free (-)-gossypol. The nanoparticles induced apoptotic morphological changes on the PC-3 cells, downregulating the mRNA expression level of Bcl-2 and upregulating the mRNA expression level of Bak. Blank nanoparticles exhibited no evident toxicity on PC-3 and RWPE-1 cells at a high dose. Therefore, the mPEG-Mal loaded (-)-gossypol nanoparticles demonstrated a favorable antitumor activity and no toxicity. The nanoparticles were able to induce the apoptosis of prostate cancer cells; thus, may be a potential antitumor nanodrug.

A study on the inhibitory effect of Radix Semiaquilegiae extract on human hepatoma HEPG-2 and SMMC-7721 cells.

The main objective of this paper was to investigate the extraction process of ethanol extract of Radix Semiaquilegiae, as well as its inhibitory activity on human hepatoma HepG-2 and SMMC-7721 cells, and to compare the inhibitory effects of different concentrations of ethanol extracts against these two hepatoma cells. Ethanol reflux extraction and ultrasound-assisted extraction with ethanol at room temperature were used in the extraction process, and MTT assay was mainly used in the activity experiment to perform in-vitro anti HepG-2 and SMMC-7721 cell activity screening of ethanol extract, and to calculate the cell inhibition rates of the extracts. The results showed that among the two types of extracts, ethanol reflux extract had more superior antitumour activity to that of the ultrasonic extract, but all of the extracts obtained had certain anti-cancer activities, and the anti-proliferative activity increased with the increase of concentration.