Regulation of base excision repair during adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages. It has been shown that the high-efficiency DNA-repair capacity of hMSCs is decreased during their differentiation. However, the underlying its mechanism during adipogenesis and osteogenesis is unknown. Herein, we investigated how alkyl-damage repair is modulated during adipogenic and osteogenic differentiation, especially focusing on the base excision repair (BER) pathway. Response to an alkylation agent was assessed via quantification of the double-strand break (DSB) foci and activities of BER-related enzymes during differentiation in hMSCs. Adipocytes showed high resistance against methyl methanesulfonate (MMS)-induced alkyl damage, whereas osteoblasts were more sensitive than hMSCs. During the differentiation, activities, and protein levels of uracil-DNA glycosylase were found to be regulated. In addition, ligation-related proteins, such as X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase ß, were upregulated in adipocytes, whereas their levels and recruitment declined during osteogenesis. These modulations of BER enzyme activity during differentiation influenced DNA repair efficiency and the accumulation of DSBs as repair intermediates in the nucleus. Taken together, we suggest that BER enzymatic activity is regulated in adipogenic and osteogenic differentiation and these alterations in the BER pathway led to different responses to alkyl damage from those in hMSCs.

Belvarafenib penetrates the BBB and shows potent antitumor activity in a murine melanoma brain metastasis model.

Brain metastasis is a common complication in melanoma patients with BRAF and NRAS mutations and has a poor prognosis. Although BRAF inhibitors are clinically approved, their poor brain penetration limits their efficacy in brain metastasis. Thus, melanoma brain metastasis still requires better treatment. Belvarafenib, a pan-RAF inhibitor, has reported antitumor activity in melanoma with RAF and RAS mutations in animal models and patients. However, brain permeability and antitumor efficacy on brain metastasis have not been determined. This study confirmed the brain penetration of belvarafenib, the antitumor activity on BRAF and NRAS mutant melanoma, and the efficacy on melanoma within the brain. Belvarafenib strongly suppressed melanoma in BRAF V600E mutant A375SM tumor-bearing mice. It also significantly inhibited tumor growth in NRAS mutant SK-MEL-30 and K1735 tumor-bearing mice and synergized to enhance the antitumor activity combined with cobimetinib or atezolizumab. Belvarafenib was penetrated at considerable levels into the brains of mice and rats following oral administration. The exposure of belvarafenib in the brain was similar to or higher than that in plasma, and this high brain penetration differed significantly from that of other BRAF inhibitors with low brain penetration. Most importantly, belvarafenib strongly reduced tumor burden and markedly improved survival benefits in mice intracranially implanted with A375SM melanoma. These results demonstrated that belvarafenib, which has favorable BBB permeability, and potent antitumor activity on the tumors with BRAF/NRAS mutations, may be a promising therapeutic option for patients with BRAF/NRAS mutant melanoma brain metastasis.

Synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel FMS inhibitors.

Colony stimulating factor-1 receptor (CSF-1R or FMS) and it ligand, CSF-1, signaling regulates the differentiation and function of tumor-associated macrophages (TAMs) that play an important role in tumor progression. Derivatives of thieno[3,2-d]pyrimidine were synthesized and evaluated as kinase inhibitors of FMS. The most representative compound 21 showed strong activity (IC50 = 2 nM) against FMS kinase and served as candidate for proof of concept. Anti-tumor activity alone and/or in combination with paclitaxel was examined via a tumor cell growth inhibition assay and via an in vitro tumor invasion assay using human breast adenocarcinoma cells.

Exendin-4 protects against sulfonylurea-induced ß-cell apoptosis.

Sulfonylurea is one of the commonly used anti-diabetic drugs that stimulate insulin secretion from ß-cells. Despite their glucose lowering effects in type 2 diabetes mellitus, long-term treatment brought on secondary failure characterized by ß-cell exhaustion and apoptosis. ER stress induced by Ca(2+) depletion in endoplasmic reticulum (ER) is speculated be one of the causes of secondary failure, but it remains unclear. Glucagon like peptide-1 (GLP-1) has anti-apoptotic effects in ß-cells after the induction of oxidative and ER stress. In this study, we examined the anti-apoptotic action of a GLP-1 analogue in ß-cell lines and islets against ER stress induced by chronic treatment of sulfonylurea. HIT-T15 and dispersed islet cells were exposed to glibenclamide for 48 h, and apoptosis was evaluated using Annexin/PI flow cytometry. Expression of the ER stress-related molecules and sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2/3 was determined by real-time PCR and western blot analysis. Chronic exposure to glibenclamide increased apoptosis by depletion of ER Ca(2+) concentration through reduced expression of SERCA 2/3. Pretreatment with Exendin-4 had an anti-apoptotic role through ER stress modulation and ER Ca(2+) replenishing by SERCA restoration. These findings will further the understanding of one cause of glibenclamide-induced ß-cell loss and therapeutic availability of GLP-1-based drugs in secondary failure by sulfonylurea during treatment of diabetes.

Exendin-4 Protects Against Sulfonylurea-Induced ß-Cell Apoptosis.

Sulfonylurea is one of the commonly used anti-diabetic drugs that stimulate insulin secretion from ß-cells. Despite their glucose lowering effects in type 2 diabetes mellitus, long-term treatment brought on secondary failure characterized by ß-cell exhaustion and apoptosis. ER stress induced by Ca2+ depletion in endoplasmic reticulum (ER) is speculated be one of the causes of secondary failure, but it remains unclear. Glucagon like peptide-1 (GLP-1) has anti-apoptotic effects in ß-cells after the induction of oxidative and ER stress. In this study, we examined the antiapoptotic action of a GLP-1 analogue in ß-cell lines and islets against ER stress induced by chronic treatment of sulfonylurea. HIT-T15 and dispersed islet cells were exposed to glibenclamide for 48 h, and apoptosis was evaluated using Annexin/PI flow cytometry. Expression of the ER stress-related molecules and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2/3 was determined by real-time PCR and western blot analysis. Chronic exposure to glibenclamide increased apoptosis by depletion of ER Ca2+ concentration through reduced expression of SERCA 2/3. Pretreatment with Exendin-4 had an anti-apoptotic role through ER stress modulation and ER Ca2+ replenishing by SERCA restoration. These findings will further the understanding of one cause of glibenclamide-induced ß-cell loss and therapeutic availability of GLP-1-based drugs in secondary failure by sulfonylurea during treatment of diabetes.

Anatomical reconstruction of the spring ligament using peroneus longus tendon graft.

Posterior tibial tendon insufficiency is often associated with failure of the spring ligament and flatfoot deformity. Arch correction procedures involving bony realignment, such as lateral column lengthening or joint fusions, can predispose to arthritis. Soft tissue reconstruction may provide a more anatomical correction without these complications. The purpose of this investigation was to compare the ability of three different spring ligament reconstruction procedures to correct flatfoot deformity. A deformity model of 5 degrees - 15 degrees talonavicular abduction was created in 10 cadaver foot-ankle specimens. Three reconstructions utilizing the peroneus longus tendon were evaluated for their ability to correct talonavicular abduction and subtalar eversion under 357 N vertical GRF load. A superomedial/plantar passage of the tendon through the calcaneus and navicular was shown to be more effective than either of the other two approaches, correcting the talonavicular joint from 9.1 degrees +/- 8.1 degrees abducted to 1.0 degree +/- 6.8 degrees adducted, and the subtalar joint from 3.1 degrees +/- 3.3 degrees everted to 0.4 degrees +/- 4.2 degrees inverted. Thus, an anatomical reconstruction of a model of a failed spring ligament was demonstrated to be effective in the correction of a flatfoot deformity produced in cadaver foot-ankle specimens.