PAX2 Regulates ADAM10 Expression and Mediates Anchorage-Independent Cell Growth of Melanoma Cells

PAX transcription factors play an important role during development and carcinogenesis. In this study, we investigated PAX2 protein levels in melanocytes and melanoma cells by Western Blot and immunofluorescence analysis and characterized the role of PAX2 in the pathogenesis of melanoma. In vitro we found weak PAX2 protein expression in keratinocytes and melanocytes. Compared to melanocytes increased PAX2 protein levels were detectable in melanoma cell lines. Interestingly, in tissue sections of melanoma patients nuclear PAX2 expression strongly correlated with nuclear atypia and the degree of prominent nucleoli, indicating an association of PAX2 with a more atypical cellular phenotype. In addition, with chromatin immunoprecipitation assay, PAX2 overexpression and PAX2 siRNA we present compelling evidence that PAX2 can regulate ADAM10 expression, a metalloproteinase known to play important roles in melanoma metastasis. In human tissue samples we found co-expression of PAX2 and ADAM10 in melanocytes of benign nevi and in melanoma cells of patients with malignant melanoma. Importantly, the downregulation of PAX2 by specific siRNA inhibited the anchorage independent cell growth and decreased the migratory and invasive capacity of melanoma cells. Furthermore, the downregulation of PAX2 abrogated the chemoresistance of melanoma cells against cisplatin, indicating that PAX2 expression mediates cell survival and plays important roles during melanoma progression

Study protocol:Effects of active versus passive recharge burst spinal cord stimulation on pain experience in persistent spinal pain syndrome type 2: a multicentre randomized trial (BURST-RAP study)

BACKGROUND: Spinal cord stimulation (SCS) has shown to be an effective treatment for patients with persistent spinal pain syndrome type 2 (PSPS Type 2). The method used to deliver electrical charge in SCS is important. One such method is burst stimulation. Within burst stimulation, a recharge pattern is used to prevent buildup of charge in stimulated tissues. Two variations of burst waveforms are currently in use: one that employs active recharge and one that uses passive recharge. It has been suggested that differences exist between active and passive recharge paradigms related to both efficacy of pain relief and their underlying mechanism of action. Active recharge has been shown to activate both the medial spinal pathway, engaging cortical sensorimotor areas involved in location and intensity of pain, and lateral pathway, reaching brain areas involved with cognitive-emotional aspects of pain. Passive recharge has been suggested to act via modulation of thalamic neurons, which fire in a similar electrical pattern, and thereby modulate activity in various cortical areas including those related to motivational and emotional aspects of pain. The objective of this randomized clinical trial is to assess and compare the effect of active versus passive recharge Burst SCS on a wide spectrum of pain in PSPS Type 2 patients. METHODS: This multicentre randomized clinical trial will take place in 6 Dutch hospitals. PSPS Type 2 patients (n=94) will be randomized into a group receiving either active or passive recharge burst. Following a successful trial period, patients are permanently implanted. Patients complete the Pain Catastrophizing Scale (PCS) (primary outcome at 6 months), Numeric Pain Rating Scale (NRS), Patient Vigilance and Awareness Questionnaire (PVAQ), Hospital Anxiety and Depression Scale (HADS), Quality of Life (EQ-5D), Oswestery Disability Index (ODI), Patient Global Impression of Change (PGIC) and painDETECT questionnaires (secondary outcomes) at baseline, after trial, 1, 3, 6 and 12 months following implantation. DISCUSSION: The BURST-RAP trial protocol will shed light on possible clinical differences and effectivity of pain relief, including emotional-motivational aspects between active and passive burst SCS in PSPS Type 2 patients. TRIAL REGISTRATION: ClinicalTrials.gov registration: NCT05421273 . Registered on 16 June 2022. Netherlands Trial Register NL9194. Registered on 23 January 2021

A Comparison of 1000 Hz to 30 Hz Spinal Cord Stimulation Strategies in Patients with Unilateral Neuropathic Leg Pain Due to Failed Back Surgery Syndrome: A Multicenter, Randomized, Double-Blinded, Crossover Clinical Study (HALO)

Introduction: Multicenter, randomized, double-blinded crossover study. The Netherlands (ClinicalTrials.gov NCT02112474). We hypothesized that the pain suppressive effects of 1000 Hz and 30 Hz spinal cord stimulation (SCS) strategies are equally effective in patients with chronic, neuropathic, unilateral leg pain after back surgery. Methods: Thirty-two patients (18–70 years, minimum leg pain 50 mm on 100 mm visual analog scale (VAS), minimal back pain) were randomized (1:1) to start 1000 Hz or 30 Hz neurostimulation for 9 days. After a 5-day washout, they crossed over, for another 9 days. Primary outcome was pain suppression (mean of VAS scores 4×/day) during the crossover period. The main investigators were blinded to strategy allocation, patients were blinded to the outcome, a blinded assessor analyzed the primary outcome. Results: The primary outcome was analyzed in 26 patients. There was no period effect (delta 4 mm, p = 0.42, 95% CI [− 5, 13]), allowing direct intrapatient comparison of the treatment effect (delta 1 mm, p = 0.92, 95% CI [− 13, 14]). Ninety-two percent of patients in both periods experienced greater than 34% pain suppression (minimal clinically important difference, MCID). Secondary outcomes (22 patients): pain suppression and improved quality of life were sustained at 12 months; both were statistically significant and clinically relevant. Fifty percent of patients had greater than 80% pain suppression (p < 0.001). At study termination, all events were resolved; no unanticipated events were reported. Medtronic provided a grant for additional study costs. Conclusion: We conclude that our hypothesis regarding the effect of 1000 Hz and 30 Hz stimulation strategies on pain suppression was confirmed. Both stimulation strategies led to a large, sustainable, clinically relevant pain suppression and improvement in quality of life

Spinal Cord Stimulation With Additional Peripheral Nerve/Field Stimulation vs Spinal Cord Stimulation Alone on Back Pain and Quality of Life in Patients With Failed Back Surgery Syndrome

INTRODUCTION: Failed back surgery syndrome (FBSS) refers to new or persistent pain following spinal surgery for back or leg pain in a subset of patients. Spinal cord stimulation (SCS) is a neuromodulation technique that can be considered in patients with predominant leg pain refractory to conservative treatment. Patients with predominant low back pain benefit less from SCS. Another neuromodulation technique for treatment of chronic low back pain is subcutaneous stimulation or peripheral nerve field stimulation (PNFS). We investigated the effect of SCS with additional PNFS on pain and quality of life of patients with FBSS compared with that of SCS alone after 12 months. MATERIALS AND METHODS: This is a comparative study of patients with FBSS who responded to treatment with either SCS + PNFS or SCS only following a multicenter randomized clinical trial protocol. In total, 75 patients completed the 12-month follow-up: 21 in the SCS-only group and 54 in the SCS + PNFS group. Outcome measures were pain (visual analog scale), quality of life (36-Item Short Form Survey [SF-36]), anxiety and depression (Hospital Anxiety and Depression Scale [HADS]), overall health (EuroQol Five-Dimension [EQ-5D]), disability (Oswestry Disability Index [ODI]), and pain assessed by the McGill questionnaire. RESULTS: There were no significant differences in baseline characteristics between the two groups. Both groups showed a significant reduction in back and leg pain at 12 months compared with baseline measurements. No significant differences were found between the groups in effect on both primary (pain) and secondary parameters (SF-36, HADS, EQ-5D, ODI, and McGill pain). CONCLUSION: In a subgroup of patients with chronic back and leg pain, SCS alone provided similar long-term pain relief and quality-of-life improvement as PNFS in addition to SCS. In patients with refractory low back pain not responding to SCS alone, adding PNFS should be recommended. CLINICAL TRIAL REGISTRATION: The Clinicaltrials.gov registration number for the study is NCT01776749

Immunohistochemical analysis of PAX2 expression in tissue sections of benign nevi and malignant melanoma.

<p>(<b>A</b>) In normal sweat glands, PAX2 is expressed in gland epithelial cells (black arrows) while intermingled stromal cells only show very weak or absent nuclear PAX2 expression (green arrow) Bar represent 100 µm. (<b>B, C</b>) Normal appearing epidermal cell layers adjacent to (<b>B,</b> bar represent 100 µm) nevi or (<b>C,</b> bar represent 200 µm) malignant melanoma show a differentially PAX2 expression with strongest PAX2 levels in germinal basal cell layers (black arrows) decreasing in higher differentiated keratinocytes and finally being absent in corneocytes (green arrows). (<b>D</b>) Malignant melanoma cells constantly exhibit a heterogeneous nuclear PAX2 expression. Strongest expression is observed in large atypical nuclei with prominent nucleoli (black arrows). Bar represent 50 µm. (<b>E, F</b>) PAX2 expression in intradermal nevi was heterogeneous and did not correlate with histological features (Original magnification: A–C: 20×; D–F: 40×). Bars represent 50 µm.</p

Downregulation of PAX2 decreases the proliferation, migration and invasion of melanoma cells.

<p>Anchorage-dependent (<b>A</b>) and anchorage-independent (<b>B</b>) cell growth was investigated by using a MTT proliferation assay. Twenty-four hours after siRNA transfection, SkMel5 cells treated with transfection reagents alone (mock) or transfected with scrambled siRNA (sc-siRNA) or PAX2-specific siRNAs were seeded into uncoated anchorage dependent cell growth) or polyHEME coated (anchorage independent cell growth) 96 well plates and cell growth was measured 24, 48 and 72 hours later using a MTT-assay. 3 independent experiments have been performed and statistical analysis has been performed using Anova post-hoc analysis. ***P<0.001 considered statistically significant compared to control transfected cells (Mock). <b>###</b>P<0.001 considered statistically significant compared to scrambled-siRNA transfected cells, *P<0.01 considered statistically significant compared to scrambled-siRNA transfected cells. (<b>C</b>) Migration assay of SkMel5 cells was performed 48 h after the transfection with control siRNA (sc-siRNA) or PAX2 specific siRNA (PAX2-siRNA). ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA). (<b>D</b>) The invasive capacity of SkMel5 cells was analyzed 48 h after the transfection of contol (sc-siRNA) or PAX2 siRNA (PAX2-siRNA) in an invasion assay as described under material and methods ***P<0.001 considered statistically significant compared to control siRNA transfected cells (sc-siRNA).</p