JAG-1/Notch signaling axis in the spinal cord contributes to bone cancer pain in rats.

Notch signal plays an important role in regulating cell-cell interactions with the adjacent cells. However, it remains unknown whether Jagged1 (JAG-1) mediated Notch signaling regulates bone cancer pain (BCP) via the spinal cell interactions mechanism. Here, we showed that intramedullary injection of Walker 256 breast cancer cells increased the expression of JAG-1 in spinal astrocytes and knockdown of JAG-1 reduced BCP. The supplementation of exogenous JAG-1 to the spinal cord induced BCP-like behavior and promoted expression of c-Fos and hairy and enhancer of split homolog-1 (Hes-1) in the spinal cord of the naïve rats. These effects were reversed when the rats were administered intrathecal injections of N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The intrathecal injection of DAPT reduced BCP and inhibited Hes-1 and c-Fos expression in the spinal cord. Furthermore, our results showed that JAG-1 up-regulated Hes-1 expression by inducing the recruitment of Notch intracellular domain (NICD) to the RBP-J/CSL-binding site located within the Hes-1 promoter sequence. Finally, the intrathecal injection of c-Fos-antisense oligonucleotides (c-Fos-ASO) and administration of sh-Hes-1 to the spinal dorsal horn also alleviated BCP. The study indicates that inhibition of the JAG-1/Notch signaling axis may be a potential strategy for the treatment of BCP.

CXCR1 participates in bone cancer pain induced by Walker 256 breast cancer cells in female rats.

Bone cancer pain (BCP) is a clinically intractable mixed pain, involving inflammation and neuropathic pain, and its mechanisms remain unclear. CXC chemokine receptor 1 (CXCR1, IL-8RA) and 2 (CXCR2, IL-8RB) are high-affinity receptors for interleukin 8 (IL8). According to previous studies, CXCR2 plays a crucial role in BCP between astrocytes and neurons, while the role of CXCR1 remains unclear. The objective of this study was to investigate the role of CXCR1 in BCP. We found that CXCR1 expression increased in the spinal dorsal horn. Intrathecal injection of CXCR1 siRNA effectively attenuated mechanical allodynia and pain-related behaviors in rats. It was found that CXCR1 was predominantly co-localized with neurons. Intrathecal injection of CXCR1-siRNA reduced phosphorylated JAK2/STAT3 protein levels and the NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß) levels. Furthermore, in vitro cytological experiments confirmed this conclusion. The study results suggest that the spinal chemokine receptor CXCR1 activation mediates BCP through JAK2/STAT3 signaling pathway and NLRP3 inflammasome (NLRP3, caspase1, and IL-1ß).

miR-155-5p in the spinal cord regulates hypersensitivity in a rat model of bone cancer pain.

BACKGROUND: Noncoding microRNAs have emerged as critical players of gene expression in the nervous system, where they contribute to regulating nervous disease. As stated in previous research, the miR-155-5p upregulation happens in the spinal cord at the nociceptive state. It was unclear if miR-155-5p is linked to bone cancer pain (BCP). Herein, we aimed at investigating the miR-155-5p functional regulatory function in BCP process and delineating the underlying mechanism. METHODS: The miRNA-155-5p levels and cellular distribution were determined by RNA sequencing, fluorescent in situ hybridization (FISH), and quantitative real-time PCR (qPCR). Immunoblotting, qPCR, dual-luciferase reporter gene assays, immunofluorescence, recombinant overexpression adeno-associated virus, small interfering RNA, intraspinal administration, and behavioral tests were utilized for exploring the downstream signaling pathway. RESULTS: The miR-155-5p high expression in spinal neurons contributes to BCP maintenance. The miR-155-5p blockage via the intrathecal injection of miR-155-5p antagomir alleviated the pain behavior; in contrast, upregulating miR-155-5p by agomir induced pain hypersensitivity. The miR-155-5p bounds directly to TCF4 mRNA's 3' UTR. BCP significantly reduced protein expression of TCF4 versus the Sham group. The miR-155-5p inhibition relieved the spinal TCF4 protein's down-expression level, while miR-155-5p upregulation by miR-155-5p agomir intrathecal injection decreased TCF4 protein expression in naïve rats. Additionally, TCF4 overexpression in BCP rats could increase Kv1.1. Moreover, TCF4 knockdown inhibited Kv1.1 expression in BCP rats. Indeed, TCF4 and Kv1.1 were co-expressed in BCP spinal cord neurons. CONCLUSION: The study findings stated the miR-155-5p pivotal role in regulating BCP by directly targeting TCF4 in spinal neurons and suggested that miR-155-5p could be a promising target in treating BCP.

Macrophage renewal modes affect acquired resistance to gefitinib in EGFR­mutant lung cancer PC­9 cells.

EGFR tyrosine kinase inhibitors (TKIs) have been successfully used in lung cancer treatment. Nevertheless, tumor cells may develop resistant phenotypes in the tumor microenvironment after a period of treatment with any generation of EGFR TKIs. Macrophages play a pivotal role in carcinogenesis and development. At the same time, macrophages renew continuously, and their polarized phenotype changes dynamically. Although macrophage polarization is generally considered to be involved in secondary resistance, it would be time consuming if renewed macrophages would only protect tumor cells from drug attacks after polarization. It is unclear how dynamic renewal of macrophage contributes to acquired resistance. By using two co­culture approaches, the present study modeled two types of renewal of macrophages: Migrated macrophages (Mm) and locally self­renewal resident macrophages (Mr). The EGFR­mutant lung cancer cell line PC­9 was induced to develop resistance to gefitinib in these co­culture approaches and without macrophages co­culture. PC­9 cells induced in Mm co­culture recovered fast from gefitinib attack, and formed the gefitinib resistant (GR) subline PC­9/Mm/GR with the strongest resistant ability. PC­9/Mm/GR cells exhibited epithelial characteristics, including stable expression of EGFR, phosphorylated EGFR, insulin­like growth factor 1 receptor, TGF­ßII receptor and E­cadherin. PC­9 cells induced in Mr co­culture and those without co­culture developed similar weak resistance to gefitinib. TGF­ß secretion was inhibited when PC­9 cells were in Mr co­culture, but not in Mm co­culture. Macrophages were not polarized in either co­culture type. The present findings suggested that macrophages may affect resistance acquirement by different renewal modes. Mm could promote resistance acquirement by stabilizing tumor cell phenotypes, which would happen before polarization. It would be helpful to monitor the response to EGFR­TKIs and design novel treatment strategies if macrophages from two renewal modes were distinguished effectively.

Upregulation of LncRNA71132 in the spinal cord regulates hypersensitivity in a rat model of bone cancer pain.

ABSTRACT: Bone cancer pain (BCP) is a pervasive clinical symptom which impairs the quality life. Long noncoding RNAs (lncRNAs) are enriched in the central nervous system and play indispensable roles in numerous biological processes, while its regulatory function in nociceptive information processing remains elusive. Here, we reported that functional modulatory role of ENSRNOT00000071132 (lncRNA71132) in the BCP process and sponging with miR-143 and its downstream GPR85-dependent signaling cascade. Spinal lncRNA71132 was remarkably increased in the rat model of bone cancer pain. The knockdown of spinal lncRNA71132 reverted BCP behaviors and spinal c-Fos neuronal sensitization. Overexpression of spinal lncRNA71132 in naive rat generated pain behaviors, which were accompanied by increased spinal c-Fos neuronal sensitization. Furthermore, it was found that lncRNA71132 participates in the modulation of BCP by inversely regulating the processing of miR-143-5p. In addition, an increase in expression of spinal lncRNA71132 resulted in the decrease in expression of miR-143 under the BCP state. Finally, it was found that miR-143-5p regulates pain behaviors by targeting GPR85. Overexpression of miR-143-5p in the spinal cord reverted the nociceptive behaviors triggered by BCP, accompanied by a decrease in expression of spinal GPR85 protein, but no influence on expression of gpr85 mRNA. The findings of this study indicate that lncRNA71132 works as a miRNA sponge in miR-143-5p-mediated posttranscriptional modulation of GPR85 expression in BCP. Therefore, epigenetic interventions against lncRNA71132 may potentially work as novel treatment avenues in treating nociceptive hypersensitivity triggered by bone cancer.

Risk Factors Affecting the Outcomes of CT-Guided Radiofrequency Thermocoagulation of Thoracic Sympathetic Nerve in the Treatment of Primary Palm Hyperhidrosis.

BACKGROUND: Primary palm hyperhidrosis (PPH) is a chronic disease characterized by uncontrolled palm-sweating exceeding physiological needs. It negatively impacts the quality of life of the patients and can lead to different degrees of psychological problems. Currently, there are a variety of treatment options for PPH, of which thoracotomy is a first-line treatment that has shown good efficacy. However, since it is an invasive procedure requiring general anesthesia and is often associated with high costs and serious complications, better alternatives should be explored. Computed tomography (CT)-guided percutaneous puncture of radiofrequency thermocoagulation (RF-TC) of the thoracic sympathetic nerve is a promising alternative treatment. It is a minimally invasive procedure that can be performed under local anesthesia and is associated with rapid recovery. However, the factors affecting the duration of the surgery-related benefits and outcomes of CT-guided percutaneous RF-TC of the thoracic sympathetic nerve are unclear. OBJECTIVES: To investigate the factors influencing the outcomes of CT-guided percutaneous RF-TC of the thoracic sympathetic nerve in patients with PPH. STUDY DESIGN: A retrospective study. SETTING: This study was conducted at the Pain Department of Jiaxing University Affiliated Hospital (Jiaxing, China). METHODS: After approval by the Ethics Committee of the Affiliated Hospital of Jiaxing College, the data of 232 corresponding patients were assessed. The Kaplan-Meier method was used for survival analysis. Univariate and multivariate analyses were performed to identify factors associated with PPH and to construct a nomogram for predicting postoperative recurrence. Time-independent receiver operating characteristic (ROC) curve analyses were performed to assess the nomogram's predictive capacity. RESULTS: In the one-year survival analysis model, gender (HR = 1.573, 95%CI: 0.844 to 2.934), age (HR = 0.965, 95%CI: 0.915 to 1.018), disease course (HR = 0.960, 95%CI: 0.908 to 1.015), palm temperature difference (HR = 0.377, 95%CI: 0.287 to 0.495), perfusion index difference (HR = 0.590, 95%CI: 0.513 to 0.680) and hyperhidrosis disease severity scale (HR = 1.963, 95%CI: 0.769 to 5.011) were identified as statistically significant factors in univariate analysis, while palm temperature difference (HR = 0.589, 95%CI: 0.369 to 0.941) and perfusion index difference (HR = 0.357, 95%CI: 0.588 to 0.968) were the independent factors in the multivariate Cox proportional hazards risk model. In the 2-year survival analysis model, palm temperature difference (HR = 0.353, 95%CI: 0.261 to 0.478), perfusion index difference (HR = 0.589, 95%CI: 0.510 to 0.680) and hyperhidrosis disease severity scale (HR = 1.964, 95%CI: 0.771 to 5.006) were the statistically significant factors while palm temperature difference (HR = 0.507, 95%CI: 0.321 to 0.799) and perfusion index difference (HR = 0.789, 95%CI: 0.625 to 0.995) were the independent factors. LIMITATIONS: This single-center retrospective study was limited by its small sample size, short follow-up time, and the possibility of bias resulting from the non-random patient selection. CONCLUSION: Palm temperature difference and perfusion index difference were independent risk factors associated with prolonging the surgical benefits and reducing postoperative recurrence of CT-guided RF-TC of the sympathetic nerves in patients with PPH.

Catalpol ameliorates CFA-induced inflammatory pain by targeting spinal cord and peripheral inflammation.

Chronic, inflammatory pain is an international health concern that severely diminishes individuals' quality of life. Catalpol is an iridoid glycoside derived from the roots of Rehmannia glutinosa that possesses anti-inflammatory, antioxidant, and neuroprotective properties for the treating multiple kinds of disorders. Nevertheless, catalpol's impacts on inflammatory pain and its potential methods of action are still unclear. The purpose of this investigation is to determine the mechanism of catalpol to reduce the inflammatory pain behaviors in a rat model with complete Freund's adjuvant (CFA). Catwalk, Von-Frey, and open field testing were performed for behavioral assessment. Western blot analysis and real-time quantitative PCR (RT-PCR) were employed to identify variations in molecular expression, while immunofluorescence was utilized to identify cellular localization. Catalpol effectively reduced CFA-induced mechanical allodynia and thermal hyperalgesia when injected intrathecally. Moreover, catalpol can regulate the HDAC4/PPAR-γ-signaling pathway in CFA rat spinal cord neurons. Meanwhile catalpol significantly decreased the expression of the NF-κB/NLRP3 inflammatory axis in the spinal cord of CFA rats. In addition, both in vivo and in vitro research revealed that catalpol treatment inhibited astrocyte activation and increase inflammatory factor expression. Interestingly, we also found that catalpol could alleviate peripheral pain by inhibiting tissue inflammation. Taken together, the findings declared that catalpol may inhibit inflammatory pain in CFA rats by targeting spinal cord and peripheral inflammation.

[Effect of Bmi-1 Expression on Chemotherapy Sensitivity in THP-1 Cells].

OBJECTIVE: To investigate the effect of Bmi-1 expression on the chemosensitivity of THP-1 cells and its relative mechanism. METHODS: The pGenesil-2-Bmi-1 1 siRNA, p-MSCV-Bmi-1 plasmid was transfected into THP-1 cells to reduce or increase the expression of Bmi-1. The expression of Bmi-1 mRNA and protein was verified by PCR and Western blot. The effect of camptothecin (CPT) on the proliferation and chemosensitivity of THP-1 cells affected by Bmi-1 gene were detected by MTT assay. The expression of DNA double-strand breaks marker-γ-H2AX was detected by immunofluorescence assay. Mitochondrial membrane potential and apoptosis were observed by flow cytometry. The expression of Cytochrome C, Caspase 3, Bax and BCL-2 was detected by Western blot. RESULTS: Silencing Bmi-1 could inhibit proliferation and enhance the sensitivity of THP-1 cells to CPT, while overexpressed Bmi-1 could promote the cell proliferation and attenucate sensitivity of THP-1 cells to CPT. Silencing Bmi-1 could enhance CPT-induced DNA double-strand breaks, decrease mitochondrial membrane potential and promote CPT-induced apoptosis. While increasing Bmi-1 gene expression could attenuate CPT-induced DNA double-strand breaks, enhamce mitochondrial membrane potential and significantly reduce CPT-induced apoptosis of cells. CONCLUSION: Bmi-1 expression could influence the sensitivity of THP-1 cells to CPT, and its relative mechanism may relate to DNA double-strand breaks and endogenous apoptotic pathways.

Sulforaphane alleviates hyperalgesia and enhances analgesic potency of morphine in rats with cancer-induced bone pain.

Due to the efficacy and tolerability of the available drugs, the current treatment for cancer-induced bone pain (CIBP) is not considered ideal, and new drugs are required for better treatment results. This study investigated whether intrathecal injection of sulforaphane (SFN) can modulates the noxious behavior associated with CIBP and enhances the analgesic effects of morphine and the possible mechanisms related to these effects were investigated. Walker256 breast cancer cells were injected into the bone marrow cavity of rats to establish the CIBP model. When CIBP rats began to exhibit painful behavior (CIBP 6 days), SFN was injected intrathecally for 7 days. The results showed that SFN alleviated the painful behavioral hypersensitivity caused by cancer, accompanied by nuclear factor, erythroid 2 like 2 (Nrf2), Haem oxygenase 1 (HO-1) activation, nuclear factor kappa B (NF-κB) inhibition and inflammation-related factors (tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-ß), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS) reduction. In addition, SFN treatment inhibited the proliferation of Walker 256 cells in a dose-dependent manner, promoted mu-opioid receptor (MOR) expression in SH-SY5Y cells and enhanced the antihyperalgesic effects of morphine on CIBP rats by restoring the downregulation of MOR expression in the spinal cord. Interestingly, the antihyperalgesic effects of SFN were partially blocked by opioid receptor antagonists. This study showed that SFN combined with morphine might be a new way to treat CIBP.

Rosiglitazone Alleviates Mechanical Allodynia of Rats with Bone Cancer Pain through the Activation of PPAR-γ to Inhibit the NF-κB/NLRP3 Inflammatory Axis in Spinal Cord Neurons.

Bone cancer pain (BCP) is a serious clinical problem that affects the quality of life of cancer patients. However, the current treatment methods for this condition are still unsatisfactory. This study investigated whether intrathecal injection of rosiglitazone modulates the noxious behaviors associated with BCP, and the possible mechanisms related to this effect were explored. We found that rosiglitazone treatment relieved bone cancer-induced mechanical hyperalgesia in a dose-dependent manner, promoted the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in spinal cord neurons, and inhibited the activation of the nuclear factor-kappa B (NF-κB)/nod-like receptor protein 3 (NLRP3) inflammatory axis induced by BCP. However, concurrent administration of the PPAR-γ antagonist GW9662 reversed these effects. The results show that rosiglitazone inhibits the NF-κB/NLRP3 inflammation axis by activating PPAR-γ in spinal neurons, thereby alleviating BCP. Therefore, the PPAR-γ/NF-κB/NLRP3 signaling pathway may be a potential target for the treatment of BCP in the future.

[Effect of Bmi-1 on Multidrug Resistance in K562/ADR Cells and Its Mechanisms].

OBJECTIVE: To investigate the effect of Bmi-1 gene silencing on drug resistance of leukemia cell K562/ADR and to explore its possible mechanism. METHODS: After two sequences of Bmi-1-siRNA were transfected into drug-resistant K562/ADR cells, the mRNA and protein expressions of Bmi-1 gene were detected. After Bmi-1 gene silencing the expression of P-gp and MDR1 were detected and the accumulation of doxorubicin in K562/ADR cells were detected by flow cytometry to determine the effect of Bmi-1 gene silencing on drug resistance of K562/ADR cells. The protein expression of NF-κB was analyzed after Bmi-1 gene silencing. Then after K562/ADR cells were treated with NF-κB inhibitor PDTC, the protein expression of P-gp and its functional changes were analyzed to determine the effect of NF-κB on drug resistance of leukemia cells. The protein expressions of PTEN, AKT and p-AKT after Bmi-1 gene silencing were detected and the effect of Bmi-1 gene silencing on PTEN/PI3K/AKT signaling pathway in drug-resistant cells was determined. After K562/ADR cells were treated with PI3K/AKT pathway inhibitor LY294002, the protein expressions of NF-κB and P-gp were analyzed to determine the regulation of AKT on the expression of NF-κB and P-gp. The protein expressions of AKT, p-AKT, NF-κB and P-gp were detected after the Bmi-1-siRNA transfected cells were treated by PTEN inhibitor BPV. Above-mentioned expression of mRNA was detected by RT-PCR, and the protein expression was detected by Western blot. RESULTS: The expression of Bmi-1 gene in K562/ADR cells decreased at both mRNA and protein levels and the doxorubicin accumulation increased after Bmi-1 gene silencing. The expression of MDR1/P-gp in Bmi-1-siRNA transfected cells was lower than that in K562/ADR cells (P＜0.05). After Bmi-1 gene silencing, the activity of NF-κB decreased. The activity of NF-κB and P-gp expression was inhibited and the function of P-gp in K562/ADR cells was reduced by using NF-κB inhibitor (PDTC). The protein expression of PTEN increased while the protein expression of p-AKT decreased after Bmi-1 gene silencing (P＜0.05). The protein expressions of p-AKT, P-gp and the activity of NF-κB were inhibited significantly by using PI3K/AKT inhibitor LY294002 (P＜0.05). After the Bmi-1-siRNA transfected cells were treated by PTEN inhibitor BPV, the activity of NF-κB and the protein expressions of P-gp were restored. CONCLUSION: Bmi-1 plays a key role in MDR-mediated multidrug resistance in K562/ADR cells, which may be mediated by activating PTEN/AKT pathway to regulate NF-κB.

[Effect of Bmi-1 Silencing on the Proliferation of K562/ADM Cells in vitro and in vivo].

OBJECTIVE: To investigate the effect of Bmi-1 gene silencing on the proliferation of K562/ADM cells in vitro and in vivo and to explore its possible molecular mechanism. METHODS: The small interference RNA (siRNA) sequences of Bmi-1 were transfected into K562/ADM cells for decreasing the expression of Bmi-1. The effect of Bmi-1 silencing on the proliferation of K562/ADM cells in vitro and in vivo was detected by using MTT method, cell colony-forming test and tumoriganicity of nude mice; the expression of Bmi-1, PTEN and PAKT proteins was detected by Wertern blot. The immunohistochemistry assay was used to analyze the expression of Bmi-1 and Ki-67. RESULTS: The Bmi-1 silencing could significantly inhibit the proliferation activity of K562/ADM cells, the cell colony-forming ability and tumorigenicity were reduced. LY294002 decreased p-AKT expression, cell colony-forming ability and tumorigenicity. Bpv reduced the PTEN expression but increased p-AKT expression and restored the colony-forming ability and tumorigenesis of K562/ADM-S1-Bpv cells. Bmi-1-siRNA dramatically suppressed the Bmi-1 and Ki-67 protein levels in xenograft tumor tissue. CONCLUSION: Bmi-1 can mediate the proliferation of K562/ADM cell, the PTEN/p-AKT might be involved in this pathway.

[Bmi-1-siRNA Regulates the Proliferation of K562 Leukemia Cells in vitro and in vivo by PTEN/pAKT Pathway].

OBJECTIVE: To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. METHODS: The Bmi-1 small interference RNA (siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. RESULTS: The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002 (an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv (an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. CONCLUSION: The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.

Grafting epoxy-modified hydrophilic polymers onto poly(dimethylsiloxane) microfluidic chip to resist nonspecific protein adsorption.

In order to achieve a simple covalent hydrophilic polymer coating on poly(dimethylsiloxane) (PDMS) microfluidic chip, epoxy modified hydrophilic polymers were synthesized in aqueous solution with a persulfate radical initiation system, and crosslinked onto PDMS pretreated by oxygen plasma and silanized with 3-aminopropyl-triethoxysilanes (APTES). Glycidyl methacrylate (GMA) was copolymerized with acrylamide (poly(AAM-co-GMA)) or dimethylacrylamide (poly(DAM-co-GMA)), and graft polymerized with polyvinylpyrrolidone (PVP-g-GMA) or polyvinylalcohol (PVA-g-GMA). The epoxy groups in the polymers were determined by UV spectra after derivation with benzylamine. Reflection absorption infrared spectroscopy (RAIRS) confirmed covalent grafting of GMA-modified polymers onto PDMS surface. Electroosmotic flow (EOF) in the polymer grafted microchannel was strongly suppressed within the range pH 3-11. Surface adsorption of lysozyme and bovine serum albumin (BSA) was reduced to less than 10% relative to that on the native PDMS surface. On the GMA-modified polymer coated PDMS microchip, basic proteins, peptides, and sodium dodecyl sulfate (SDS) denatured proteins were separated successfully.

Establishment and biological characteristics of acquired gefitinib resistance in cell line NCI-H1975/gefinitib-resistant with epidermal growth factor receptor T790M mutation.

Non­small cell lung cancer (NSCLC) cells harboring mutations in the epidermal growth factor receptor (EGFR) gene initially respond well to EGFR tyrosine kinase inhibitors (TKI), including gefitinib. However the tumor cells will invariably develop acquired resistance to the drug. The EGFR T790M mutation is generally considered to be the molecular genetic basis of acquired TKI resistance. The present study aimed to explore how the T790M mutation induces tumor cells to escape inhibition by TKI treatment. An acquired gefitinib­resistant cell line (NCI­H1975/GR) was generated from the NCI­H1975 human NSCLC cell line, which harbors the sensitive L858R and resistant T790M mutations of EGFR. The resistant cell line was established by exposing the cells intermittently to increasing concentrations of gefitinib. The mechanisms by which NSCLC acquires resistance to TKIs based on the T790M mutation, were investigated by detecting the protein expression levels of the EGFR/Kirsten rat sarcoma viral oncogene homolog (KRAS)/v­Raf murine sarcoma viral oncogene homolog B (BRAF) transduction pathway, and epithelial­mesenchymal transition (EMT) with immunocytochemistry. The resistance of the NCI­H1975/GR cells to gefitinib was 2.009­fold, as compared with the parent cells; however, the protein expression levels of EGFR, KRAS and BRAF were lower in the resistant cells. Some mesenchymal morphology was observed in the NCI­H1975/GR cells, alongside a decreasing E­cadherin expression and increasing vimentin expression. These results suggest that the reactivation of the EGFR/KRAS/BRAF transduction pathway was not detected in the NCI­H1975/GR cells. EMT may have an important role in the development of acquired resistance to EGFR­TKIs in NSCLC cells with sensitivity and resistance mutations.

The reverse effect of X-ray irradiation on acquired gefitinib resistance in non-small cell lung cancer cell line NCI-H1975 in vitro.

The clinical efficacy of gefitinib in the treatment of non-small cell lung cancer (NSCLC) with mutations in exon 18, 19 or 21 of epidermal growth factor receptor (EGFR) is limited by the acquired resistance to the drug. To explore whether X-ray irradiation could reverse the acquired gefitinib resistance in NSCLC cell in vitro. We chose a human NSCLC cell line NCI-H1975 to establish acquired gefitinib-resistant cell line named as NCI-H1975/GR. NCI-H1975/GR was irradiated with X-ray and then named as NCI-H1975/GR/XR. In the three cell lines, subsequently cell growth curves and cell population doubling time were calculated by cell proliferation assay, the changes of cell viability were evaluated by trypan blue dye exclusion method and MTT assay, the cell cycle distribution and apoptosis were investigated by flow cytometry, the expressions of E-cadherin and vimentin used to indicate epithelial-mesenchymal transition (EMT) were determined by western blot analysis, the protein expressions in EGFR/KRAS/BRAF transduction pathway were detected by immunocytochemistry, and the mutations of EGFR, KRAS and BRAF were detected by high resolution melting analysis and direct sequencing. We found that the X-ray irradiation enhanced the growth inhibitory effects of gefitinib on the acquired gefitinib-resistant cell line. Of NCI-H1975/GR/XR following gefitinib treatment, the IC50 decreased significantly, the cell proportion of phase G0/G1 was slightly higher, and the apoptosis cell proportion was significantly higher than those of NCI-H1975/GR. In addition, the reversal of EMT being present in NCI-H1975/GR cells was likely appearing in NCI-H1975/GR/XR cells. These results indicated that the acquired gefitinib resistance could be reversed by X-ray irradiation in NSCLC cell line NCI-H1975 harboring both the L858R and T790M mutation in vitro.

shRNA-mediated knockdown of Bmi-1 inhibit lung adenocarcinoma cell migration and metastasis.

Bmi-1 has been implicated in cancer cell growth and metastasis in a variety of tumor types. In this study, we sought to evaluate the expression of Bmi-1 in lung adenocarcinoma samples, and to determine if a correlation exists between Bmi-1 expression and clinical features of lung cancer, such as metastasis. Our results showed that Bmi-1 expression is increased in lung cancer tissues compared to adjacent non-cancerous tissues, and is associated with clinical features of lung cancer, including clinical stage and distant metastasis. We were then interested in determining if shRNA-mediated knockdown of Bmi-1 would inhibit metastasis of lung adenocarcinoma cells. To this end, we chose the most efficient shRNA duplexes targeting Bmi-1, and constructed two stably transfected lung adenocarcinoma cell lines (A549 and SPCA1). The shRNA-mediated knockdown of Bmi-1 significantly reduced migration in vitro, and metastasis in vivo, of A549 and SPCA1 cells. More importantly, knockdown of Bmi-1 also upregulated PTEN expression, and downregulated p-Akt and VEGF expression. These data support the hypothesis that Bmi-1 regulates key pathways involved in the metastasis of lung adenocarcinoma cells.