Differential Gene Expression Common to Acquired and Intrinsic Resistance to BRAF Inhibitor Revealed by RNA-Seq Analysis

Melanoma cells have been shown to respond to BRAF inhibitors; however, intrinsic and acquired resistance limits their clinical application. In this study, we performed RNA-Seq analysis with BRAF inhibitor-sensitive (A375P) and

Upregulation of MicroRNA-1246 Is Associated with BRAF Inhibitor Resistance in Melanoma Cells with Mutant BRAF / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Intrinsic and acquired resistance limit the therapeutic benefits of inhibitors of oncogenic BRAF in melanoma. To identify microRNAs (miRNAs) associated with resistance to a BRAF inhibitor, we compared miRNA expression levels in three cell lines with different BRAF inhibitor sensitivity. MATERIALS AND METHODS: miRNA microarray analysis was conducted to compare miRNA expression levels. Real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to confirm the expression of differentially expressed miRNAs. The cellular effects of miR-1246 were further examined by MTT assay, immunoblotting analysis, cell cycle analysis, flow cytometric assay of apoptosis, and autophagy assay. RESULTS: The miRNA microarray analysis and qRT-PCR identified five miRNAs (miR-3617, miR-92a-1, miR-1246, miR-193b-3p, and miR-17-3p) with expression that was consistently altered in two BRAF inhibitor-resistant cell lines. Among the five miRNAs, a miR-1246 mimic significantly reduced the antiproliferative effects of the BRAF inhibitor PLX4720 in BRAF inhibitor–resistant A375P (A375P/Mdr) cells, suggesting that miR-1246 upregulation confers acquired resistance to BRAF inhibition. In particular, apoptosis was identified as a major type of cell death in miR-1246–transfected cells; however, necrosis predominated in mimic-control-transfected cells, indicating that the resistance to PLX4720 in miR-1246 mimic-transfected cells is predominantly due to a reduction in necrosis. Furthermore, we found that miR-1246 promoted G2/M arrest through autophagy as a way to escape cell death by necrosis and apoptosis in response to PLX4720. The promotion of BRAF inhibitor resistance by miR-1246 was associated with lowered levels of p-ERK. CONCLUSION: These results suggest that miR-1246 may be a potential therapeutic target in melanoma with acquired resistance to BRAF inhibitors.

Induction of Resistance to BRAF Inhibitor Is Associated with the Inability of Spry2 to Inhibit BRAF-V600E Activity in BRAF Mutant Cells

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

Autophagy-Dependent Survival of Mutant B-Raf Melanoma Cells Selected for Resistance to Apoptosis Induced by Inhibitors against Oncogenic B-Raf

Most patients with mutant B-Raf melanomas respond to inhibitors of oncogenic B-Raf but resistance eventually emerges. To better understand the mechanisms that determine the long-term responses of mutant B-Raf melanoma cells to B-Raf inhibitor, we used chronic selection to establish B-Raf (V600E) melanoma clones with acquired resistance to the new oncogenic B-Raf inhibitor UI-152. Whereas the parental A375P cells were highly sensitive to UI-152 (IC5020 microM). Immunofluorescence analysis indicated the absence of an increase in the levels of P-glycoprotein multidrug resistance (MDR) transporter in A375P/Mdr cells, suggesting that resistance was not attributable to P-glycoprotein overexpression. In UI-152-sensitive A375P cells, the anti-proliferative activity of UI-152 appeared to be due to cell-cycle arrest at G0/G1 with the induction of apoptosis. However, we found that A375P/Mdr cells were resistant to the apoptosis induced by UI-152. Interestingly, UI-152 preferentially induced autophagy in A375P/Mdr cells but not in A375P cells, as determined by GFP-LC3 puncta/cell counts. Further, autophagy inhibition with 3-methyladenine (3-MA) partially augmented growth inhibition of A375P/Mdr cells by UI-152, which implies that a high level of autophagy may protect UI-152-treated cells from undergoing growth inhibition. Together, our data implicate high rates of autophagy as a key mechanism of acquired resistance to the oncogenic B-Raf inhibitor, in support of clinical studies in which combination therapy with autophagy targeted drugs is being designed to overcome resistance.

The Difference in Biological Properties between Parental and v-Ha-ras Transformed NIH3T3 Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: We performed experiments to investigate the change in cellular signaling that occurs during the transformation of a normal cell to a cell capable of cancerous growth, and we did so by using the NIH 3T3 cells that were transformed by transfection with the v-Ha-ras oncogene. MATERIALS AND METHODS: Parental and v-Ha-ras transfected NIH 3T3 cells were chosen as test systems. The siRNA transfections were performed using Lipofectamine 2000. The cell proliferation reagent WST-1 was used for the quantitative determination of cellular proliferation. Immunoblot analysis was performed using the ECL-Plus chemiluminescent system and a KODAK Image Station 4000R. RESULTS: The v-Ha-ras-transformed cells were found to be significantly more resistant to PP2 treatment, which is a potent inhibitor of the Src family tyrosine kinases, than were the parental cells at earlier times after treatment. However, PP2 induced growth arrest and the senescence-like phenotypes in both cell lines after longer treatment. Furthermore, the Raf-1 kinase of the v-Ha-ras-transformed cells was not affected by the expressed level of Sprouty proteins, which are negative regulators of the MAPK pathway, as evidenced by the failure of siRNA-mediated knockdown of Spry4 to activate Raf-1 kinase. Dephostatin (a tyrosine phosphatase inhibitor) effectively inhibited the proliferation of the v-Ha-ras transformed cells, whereas dephostatin had only a small effect on the parental cells' proliferation. This implied an inhibitory role for tyrosine phosphatase that is specific to the signaling pathway in the v-Ha-ras transformed cells. CONCLUSION: Taken together, our results show that the sustained activation of the oncogenic pathways through their resistance to negative feedback regulation might contribute to the transformation of NIH 3T3 cells.

Epidemiology of Erectile Dysfunction in one Korean Rural Community / 신경정신의학

OBJECTIVES: The purpose of this study was to investigate the prevalence of erectile dysfunction (ED) in a rural area of Korea and to examine the relationship between the prevalence and correlates of subjects. METHOD: This study was done as a part of the chronic disease survey of rural area by the Asan Foundation. Among 30 years or older population from Jung Eup, a rural area of Korea, 711 male subjects were sampled using a cluster probability sampling method. Trained interviewers visited and interviewed subjects aged 30 years or older with a structured interview. ED was categorized as 'none', 'mild', 'moderate', or 'severe' according to the ability to 'attain and/or maintain an erection satisfactory for sexual intercourse'. Response rate was 54.3% (N=386). RESULTS: The age weighted prevalence of ED was 27.2% (minimal 14.1%, moderate 7.6%, severe 5.5%). Prevalence and severity increased with age. Liver disease and underweight were significantly (p<0.05) associated with ED. Smoking seemed to be correlated with ED, but did not reach statistically significant level. CONCLUSION: ED was highly prevalent in the rural community of Korea. Medical, sociodemographic, and lifestyle variables associated with ED may alert physicians to patients at risk for ED and offer insight to its etiology.

A Comparison of Fusion Rates and Clinical Results Between Single-Level Cervical Corpectomy and Two-Level Discectomies and Fusion with Plating / 대한척추외과학회지

STUDY DESIGN: A retrospective study. OBJECTIVES: To compare the radiographic and clinical results of two-level discectomies and fusion with plating and a singlelevel corpectomy with plating. SUMMARY OF LITERATURE REVIEW: Previous studies of multisegment fusion have shown decreased fusion rates correlating with the number of increasing levels, and the use of anterior plate in multilevel fusions may be warranted because of the increased pseudoarthrosis rates. MATERIALS AND METHODS: A total 30 consecutive patients operated at our institutions between Oct. 1995 and Mar. 2000 were entered into this study. Twenty patients with cervical spondylosis had performed two-level discectomies with tricortical bone grafts and plating, and ten patients with cervical myelopathy had a single-level corpectomy with plating. Follow up averaged 2.4 years, radiographic and clinical follow up evaluation was performed. We assessed the radiologic value by postoperative and follow-up sagittal radiograms at monthly intervals until fusion was judged to be solid, and the clinical evaluation by Odom's criteria. RESULTS: Comparing the radiographic data between the two groups of patients, the values were not different. Of the thirty patients, no non-unions occurred in all patients. The average amount of graft collapse for patients with single-level corpectomy with plating or a two-level discectomy with plating was less than 1 mm for both groups. And, the average amount of kyphotic deformity was less than 1 degrees for both groups. The clinical results of the operations graded by Odom's criteria are no statistical significance between the two groups. (p < 0.9, chi test) CONCLUSION: There is no significant statistical differences for two-level discectomies with plating and a single-level corpectomy with plating in fusion rate and clinical results, and each methods can be used a viable alternative procedure by anatomical structure that were primarily causing the neural impingement with more reliable fusion rates.

Origins of Dorsal Root Ganglion Cells Innervating Medial, Lateral Collateral Ligaments and Patellar Tendon of the Rat Knee Joint / 대한정형외과학회잡지

PURPOSE: The present study was designed to examine the distribution and type of dorsal root ganglion (DRG) cells innervating the medial and lateral collateral ligaments and patellar tendons of the rat knee joint. MATERIALS AND METHODS: We selectively injected neural tracers (FG and HRP) into both collateral ligaments and patellar tendons and then charaterized the distribution and type of DRG cells in section. RESULTS: FG-labelled DRG cells, innervating the medial and lateral collateral ligaments, were located in L1-S3 (former) and L1-L5 (later). The HRP-labelled DRG cells innervating the medial and lateral collateral ligaments were smaller than 600 m2. FG-labelled DRG cells innervating the patellar tendons were found in L1 to S1. The HRP-labelled cells innervating the patellar tendons were variable in size (100 m2 to 3000 m2). CONCLUSION: This study indicates that the DRG origins of sensory nerves in the collateral ligaments and the patellar tendon of the rat knee joint are different. The majority of DRG cells innervating the patellar tendons are proprioceptive and mechanoceptive sensory cells. This finding suggests that the patellar ligaments have more neurological functions than the collateral ligaments.

Sleep Habits and Insomnia-associated Factors in the Urban Elderly: Demographic, Health and Psychobehavioral Correlates / 신경정신의학

OBJECTIVES: The purpose of this study is to analyze sleep habit and investigate the association between insomnia and demographic, health and psycho-behavioral symptomatology in Korean urban elderly. METHOD: A total of 723 community residents aged 65 years or older were surveyed by two stage, random cluster sampling method. The response rate was 76.2%. Interviewers performed door to door visits and administered a questionnaire about sleep, past medical history, and several scales as the Psychogeriatric Assessment Scale (PAS), the short form Quality of Life Scale (SF-36) and etc. RESULTS: The mean scores are as follows: retiring time was ten-sixteen p.m., wake-up time was five twenty two a.m., duration of sleep was seven hours and six minutes. Among the elderly, 11.6% and 14.8% of the subjects reported chronic and intermittent difficulty in sleeping, respectively. The patients with back pain and history of stroke complained of more insomnia symptoms in univariate analysis. In ANCOVA analysis, insomnia symptoms were associated with an old age, depressive symptoms in male. Bodily pain and history of dermatologic disease were additional risk factors in female. CONCLUSIONS: The data suggest insomnia is one of most common health problems among elderly Koreans living in the community. Strong positive relationship between depressive symptoms and insomnia were found. Further research is needed to explore the causal relationship between insomnia and risk factors.

Development of Korean Version of Structured Clinical Interview Schedule for DSM-IV Axis I Disorder: Interrater Reliability / 신경정신의학

OBJECTIVE: Accurate diagnosis and assessment for psychiatric disorders is crucial for research, as well as for clinical practice. Structured Clinical Interview for DSM-IV(SCID-RV) is a less time-consumimg and more accurate structured diagnostic interview form. It can be used by clinical professions and is known for a reliable diagnostic tool. Present study was conducted to develop Korean version of SCID-RV and to test the inter-rater reliability. METHOD: The authors have translated original SCID-RV into Korean, and revised in parallel with sociocultural background of Korea. Ninety patients from two psychiatric hospitals, both outpatient and inpatient, were interviewed and rated independently by three raters. RESULT: The kappa coefficients for most of illnesses, such as major depressive disorder, dysthymia, schizophrenia, alcohol abuse and dependency, anxiety disorder and eating disorder were excellent (>0.70) in the evaluation of current disorders. And the kappa coefficients for bipolar disorder, delusional disorder, agoraphobia, undifferentiated somatoform disorder, and hypochondriasis were acceptable (>0.40) in the evaluation of current disorders. In the evaluation of lifetime disorders, the concordant rates of all the diagnoses except bipolar disorder and undifferentiated somatoform disorder were excellent. Lack of hierarchy in DSM-IV allows for multiple Axis I diagnoses. Mean numbers of Axis I diagnoses per subject assigned by the three raters were 1.5-1.7. CONCLUSION: Our findings confirm that SCID-RV yields highly reliable diagnoses. SCID-RV is recommended for accurate diagnosis in clinical practice and research on psychiatric disorders.