Elucidation of the anti-tumor effect of the synthetic retinoid, CD437, in malignant melanoma

The synthetic retinoid 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene Carboxylic Acid (CD437/AHPN) has been shown to have an antiproliferative effect, as well as being a potent inducer of apoptosis, in many cancer cell lines. The mechanisms behind CD437s activities have been reported as cell-line dependent. In this master thesis, the anti-tumor effect of CD437 was elucidated in the two human malignant melanoma cell lines, FEMX1 and WM239. - CD437 was shown to have an antiproliferative effect in both cell lines. A reduction in cells relative to the control was first visually observed, and later confirmed by a cell count. As revealed by MTS, the reduction of viable cells was duration- and dosage dependent. - Treatment with CD437 caused a cell-line dependent cycle arrest, yielding an S phase- and a G1 phase arrest in FEMX1 and WM239 respectively, as demonstrated by flow cytometry. Western blot analysis revealed that the arrests were accompanied by upregulations of p53, p21 and E2F-1 protein levels. A down-regulation of cyclin D1 was found in FEMX1 cells. - Apoptosis was observed in both cell lines following treatment with CD437; Morphological features associated with apoptosis were seen in both cell lines. A cell count using trypan blue revealed an increase of dead cells after exposure to CD437. A TUNEL analysis demonstrated DNA fragmentation in WM239, but not in FEMX1. In both cell lines, cleavage of the initiator caspases 8 and -9 were observed, in addition to PARP cleavage. Cleavage of caspase 3 was found in FEMX1 cells. - An increase of the orphan nuclear receptor Nur77 was found using the western blot analysis, in treated the cells. In addition, activation of c-jun was observed in both cell lines following exposure to CD437. - Treatment with CD437 resulted in an activation of the p38/MAPK signaling pathway in both cell lines, as assessed by western blotting. Additionally, in the exposed FEMX1 cells the PI3K signaling pathway was activated. - Preliminary results suggest that an up-regulation of p53 caused by CD437 may be regulated by Nur77. The increase of p21 did not appear to be controlled by Nur77, which may suggest a p53-independent expression of p21. Malignant melanomas are notoriously resistant to chemotherapy, making the prognosis for patients with metastasis very poor. CD437 represent a potential new treatment option, however many more studies are needed to evaluate its effect both in vitro and in vivo.Master i biomedisi

High Expression of Wee1 Is Associated with Poor Disease-Free Survival in Malignant Melanoma: Potential for Targeted Therapy

Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G2/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21Cip1/WAF1 (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239WTp53, WM45.1MUTp53 and LOXWTp53, further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G1/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents

Elucidation of the anti-tumor effect of the synthetic retinoid, CD437, in malignant melanoma

The synthetic retinoid 6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene Carboxylic Acid (CD437/AHPN) has been shown to have an antiproliferative effect, as well as being a potent inducer of apoptosis, in many cancer cell lines. The mechanisms behind CD437s activities have been reported as cell-line dependent. In this master thesis, the anti-tumor effect of CD437 was elucidated in the two human malignant melanoma cell lines, FEMX1 and WM239. - CD437 was shown to have an antiproliferative effect in both cell lines. A reduction in cells relative to the control was first visually observed, and later confirmed by a cell count. As revealed by MTS, the reduction of viable cells was duration- and dosage dependent. - Treatment with CD437 caused a cell-line dependent cycle arrest, yielding an S phase- and a G1 phase arrest in FEMX1 and WM239 respectively, as demonstrated by flow cytometry. Western blot analysis revealed that the arrests were accompanied by upregulations of p53, p21 and E2F-1 protein levels. A down-regulation of cyclin D1 was found in FEMX1 cells. - Apoptosis was observed in both cell lines following treatment with CD437; Morphological features associated with apoptosis were seen in both cell lines. A cell count using trypan blue revealed an increase of dead cells after exposure to CD437. A TUNEL analysis demonstrated DNA fragmentation in WM239, but not in FEMX1. In both cell lines, cleavage of the initiator caspases 8 and -9 were observed, in addition to PARP cleavage. Cleavage of caspase 3 was found in FEMX1 cells. - An increase of the orphan nuclear receptor Nur77 was found using the western blot analysis, in treated the cells. In addition, activation of c-jun was observed in both cell lines following exposure to CD437. - Treatment with CD437 resulted in an activation of the p38/MAPK signaling pathway in both cell lines, as assessed by western blotting. Additionally, in the exposed FEMX1 cells the PI3K signaling pathway was activated. - Preliminary results suggest that an up-regulation of p53 caused by CD437 may be regulated by Nur77. The increase of p21 did not appear to be controlled by Nur77, which may suggest a p53-independent expression of p21. Malignant melanomas are notoriously resistant to chemotherapy, making the prognosis for patients with metastasis very poor. CD437 represent a potential new treatment option, however many more studies are needed to evaluate its effect both in vitro and in vivo

Adherence to subcutaneous interferon beta-1a treatment using an electronic injection device: a prospective open-label scandinavian noninterventional study (The scansmart study)

Background: Disease modifying drugs help control the course of relapsing remitting multiple sclerosis (RRMS); however, good adherence is needed for long-term outcomes. Objective: To evaluate patient adherence to treatment with subcutaneous interferon beta-1a using RebiSmart® and assess injection-site reactions and treatment satisfaction. Methods: This prospective, single-arm, open-label, noninterventional multicenter Phase IV trial included disease modifying drug-experienced mobile patients with RRMS. Adherence was measured over 12 weeks. Items 13–23, 35, 37, and 38 of the Multiple Sclerosis Treatment Concerns Questionnaire (injection-site reactions and treatment satisfaction) were recorded at 12 weeks. Results: Sixty patients were recruited (mean age 43.7 [±SD 7.9] years; 83% female; mean years since multiple sclerosis diagnosis 6.7 [SD 4.5]). Adherence data were obtained in 54 patients only due to technical problems with six devices. Over 12 weeks, 89% (n=48) of patients had ≥90% adherence to treatment. Most patients experienced mild influenza-like symptoms and injection-site reactions, and global side effects were minimal. Most patients (78%) rated the convenience as the most important aspect of the device, and most experienced no or mild pain. Conclusion: RRMS patients treated with subcutaneous interferon beta-1a, administered with RebiSmart, demonstrated generally good adherence, and the treatment was generally well tolerated

High Wee1 expression increases with tumor progression and is associated with a shorter relapse-free period.

<p>A. Wee1 expression in cultured melanocytes, benign nevi, primary- and metastatic melanoma, analyzed by immunohistochemistry. B. Melanoma patients were grouped according to Wee1 expression in their tumors (high (n = 44) or low (n = 63)). Relapse-free survival in months was estimated for both groups and presented as a Kaplan Meyer curve.</p

Transfection with siWee1 effectively shuts down Wee1 expression and reduces cell viability.

<p>A. Cells were transfected with siWee1 for 48 hours. Expressions of Wee1 and pCDK1^Tyr15 were examined by western blot analysis. α-tubulin was used as loading control. The figure is representative of at least three independent biological experiments.B and C. Cells were transfected with siWee1 (dots: 24 h, stripes: 48 h and no-pattern: 72 h). The relative amount of viable cells was estimated by MTS (B), and the relative quantity of living cells was estimated by counting trypan-excluding cells (C).</p

Transfection with siWee1 promotes DNA damages and apoptosis.

<p>A. Presence of cytoplasmic oligonucleosomes was measured by ELISA following 48 h siWee1 transfection. Induction of apoptosis shown as enrichment factor calculated as absorbance at 405 nm of siWee1 treated cells relative to siCtr treated cells. B. Protein expressions were measured by Western blot following 48 h transfection with either siCtr or siWee1. Cleavage of PARP and Caspase 3 are shown with arrows. α-tubulin was used as loading control. The figure is representative of at least three independent biological experiments.</p

Number (percentage) of melanocytic lesions expressing different levels of Wee1.

<p>Wee1 expressions in benign nevi, primary- and metastatic melanoma were estimated by immunohistochemistry, and categorized in four semi-quantitative classes according to percentage of immunoreactive tumor cells. The groups were further divided into low (<10%) and high (≥10%) expression.</p

Wee1 expression correlates with clinical parameters- and markers of tumor progression.

*<p>Low expression of Cyclin A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes1" target="_blank">[11]</a>, Ki67 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes1" target="_blank">[11]</a>, Cyclin D3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes2" target="_blank">[12]</a>, p21 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Florenes3" target="_blank">[13]</a> and p53 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038254#pone.0038254-Maelandsmo1" target="_blank">[14]</a>; defined as immunoreactivity in <5% of the tumor cells. Wee1 expression in <10% of tumor cells is defined as low.</p>†<p>Statistical significances determined by Chi-square tests.</p