Antigen-Capturing Mesoporous Silica Nanoparticles Enhance the Radiation-Induced Abscopal Effect in Murine Hepatocellular Carcinoma Hepa1-6 Models

Immunomodulation by radiotherapy (RT) is an emerging strategy for improving cancer immunotherapy. Nanomaterials have been employed as innovative tools for cancer therapy. This study aimed to investigate whether mesoporous silica nanoparticles (MSNs) enhance RT-mediated local tumor control and the abscopal effect by stimulating anti-cancer immunity. Hepa1-6 murine hepatocellular carcinoma syngeneic models and immunophenotyping with flow cytometry were used to evaluate the immune responses. When mice harboring bilateral tumors received 8 Gy of X-rays on a single tumor, the direct injection of MSNs into irradiated tumors enhanced the growth inhibition of irradiated and unirradiated contralateral tumors. MSNs enhanced RT-induced tumor infiltration of cytotoxic T cells on both sides and suppressed RT-enhanced infiltration of regulatory T cells. The administration of MSNs pre-incubated with irradiated cell-conditioned medium enhanced the anti-tumor effect of anti-PD1 compared to the as-synthesized MSNs. Intracellular uptake of MSNs activated JAWS II dendritic cells (DCs), which were consistently observed in DCs in tumor-draining lymph nodes (TDLNs). Our findings suggest that MSNs may capture tumor antigens released after RT, which is followed by DC maturation in TDLNs and infiltration of cytotoxic T cells in tumors, thereby leading to systemic tumor regression. Our results suggest that MSNs can be applied as an adjuvant for in situ cancer vaccines with RT

Antigen-Capturing Mesoporous Silica Nanoparticles Enhance the Radiation-Induced Abscopal Effect in Murine Hepatocellular Carcinoma Hepa1-6 Models

Immunomodulation by radiotherapy (RT) is an emerging strategy for improving cancer immunotherapy. Nanomaterials have been employed as innovative tools for cancer therapy. This study aimed to investigate whether mesoporous silica nanoparticles (MSNs) enhance RT-mediated local tumor control and the abscopal effect by stimulating anti-cancer immunity. Hepa1-6 murine hepatocellular carcinoma syngeneic models and immunophenotyping with flow cytometry were used to evaluate the immune responses. When mice harboring bilateral tumors received 8 Gy of X-rays on a single tumor, the direct injection of MSNs into irradiated tumors enhanced the growth inhibition of irradiated and unirradiated contralateral tumors. MSNs enhanced RT-induced tumor infiltration of cytotoxic T cells on both sides and suppressed RT-enhanced infiltration of regulatory T cells. The administration of MSNs pre-incubated with irradiated cell-conditioned medium enhanced the anti-tumor effect of anti-PD1 compared to the as-synthesized MSNs. Intracellular uptake of MSNs activated JAWS II dendritic cells (DCs), which were consistently observed in DCs in tumor-draining lymph nodes (TDLNs). Our findings suggest that MSNs may capture tumor antigens released after RT, which is followed by DC maturation in TDLNs and infiltration of cytotoxic T cells in tumors, thereby leading to systemic tumor regression. Our results suggest that MSNs can be applied as an adjuvant for in situ cancer vaccines with RT

MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.

Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH) is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway, thereby regulating expression of genes including insulin-like growth factor (IGF)-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R). In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs). MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2) were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b

Effects of methylsulfonylmethane (MSM) on the expression of growth hormone (GH) signaling-related proteins in osteoblast-like cells and MSCs.

<p>MG-63 (A) and UMR-106 (B) cells were treated with the indicated MSM concentrations for 24 h. (C) Mesenchymal stem cells were cultured in osteogenic medium with various concentrations of MSM for 21 days. (D) UMR-106 cells were left untreated or pretreated with 50 µM AG490 for 4 h then treated with MSM for 24 h. Protein extracts (20 µg) were separated by 10% SDS-PAGE, and Western blots were performed. β-actin was used as a protein loading control. (E) The relative levels of IGF-1R, GHR and Jak2 protein were determined using densitometric analysis and normalized to the amount of β-actin. This picture is representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p

Involvement of STAT5b in MSM-induced osteogenic marker genes in MSCs.

<p>(A) Bone marrow mesenchymal stem cells were cultured in the osteogenic medium at 5 days for ALP, 14 days for osteonectin (ON) and bone sialoprotein (BSP), and 21 days for osteocalcin (OCN) and osterix mRNA expression after the treatment with various concentrations (0, 10 and 20 mM) of MSM. RT-PCR was performed using the cDNA and primers for ALP, ON, BSP, OCN, osterix and 18S. Total RNA was isolated from the MSCs using an RNeasy kit. 18S was used as a control. (B) Bone marrow Mesenchymal stem cells and (C) C3H10T1/2 cells were cultured in osteogenic medium at 5 days for ALP and Runx2, 14 days for OPN and BSP, and 21 days for OCN and osterix mRNA expression after the treatment with 20 mM MSM. After culture, real-time PCR was performed. (D) Osteogenic differentiation marker genes (ALP, BSP, OCN, OPN, Osterix and Runx2) and STAT5b gene expression was analyzed at day 5, 14 and 21 after MSM treatment in C3H10T1/2 cells transfected with STAT5b siRNA or non-target siRNA. The effect of STAT5b knockdown on osteogenic marker genes was analyzed by real-time PCR. GAPDH was used as the internal control. The relative levels of mRNA were determined using densitometric analysis and normalized to the amount of GAPDH. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (*p<0.05, **p<0.01, ***p<0.001).</p

Methylsulfonylmethane (MSM)-induced binding activity of STAT5b to the insulin-like growth factor-1 receptor (IGF-1R) GAS site.

<p>(A) UMR-106 cells were cultured in serum-free MEM for 24 h and incubated with MSM (20 mM) for 12 or 24 h. Nuclear extracts (NE) were separated and blotted onto a nitrocellulose membrane, which showed an increase in the level of IGF-1R and STAT5b. Tata binding protein (TBP) was used as a nuclear protein loading control. (B) STAT5b DNA binding was detected by electrophoretic mobility shift assay. Nuclear extracts were prepared and incubated with STAT5 probe (from a part of Panomics EMSA kits). The resulting complexes were electrophoresed on a 6% non-denaturing gel. (C) The IGF-1R (−2350/+640) LUC reporter plasmid or (D) the 700 bp IGF-1-pGL2 were cotransfected into UMR 106 cells with the STAT5b expression vector, the β-galactosidase expression plasmid (pCMV β-Gal), and either empty pGL2 vector, and then incubated with 50 µM AG490 for 4 h, then treated with 20 mM MSM for 24 h. Promoter activities were expressed as luciferase normalized to β-galactosidase values. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p

Methylsulfonylmethane (MSM) enhances growth hormone (GH) signaling via Jak2/STAT5b activation in osteoblast-like cells.

<p>(A) Jak2 was immunoprecipitated with anti-Jak2 antibody from whole cell extracts of UMR 106 cells left untreated or pretreated with 30 nM GH for 2 h followed by MSM for 24 h. Jak2 precipitation and the phosphorylation status of Jak2 were analyzed by Western blotting with anti-Jak2 and anti-phosphotyrosine (4G10) antibodies. (B) The relative levels of Jak2 phosphorylation were determined using densitometric analysis and normalized to the amount of Jak2. (C) UMR-106 cells were incubated with 50 µM AG490 for 4 h, then left untreated or pretreated with 30 nM GH for 2 h followed by MSM for 24 h. STAT5b precipitation and the phosphorylation status of the precipitated STAT5b were analyzed by Western blot with anti-STAT5b and 4G10 antibodies. This picture is representative of three independent experiments. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by t-test (***p<0.001).</p

Methylsulfonylmethane (MSM) activates the expression of growth hormone (GH) signaling-related mRNA in UMR 106 cells.

<p>Total RNA was isolated from the UMR-106 cells using an RNeasy kit. The cDNA was amplified using specific primers for insulin like growth factor-1 receptor (IGF-1R), the growth hormone receptor (GHR) or 18S. 18S was used as a control. (A) UMR-106 cells were treated with the indicated concentrations of MSM for 24 h. (B) The relative levels of IGF-1R and GHR mRNA were determined using densitometric analysis and normalized to the amount of 18S. (C) UMR-106 cells were left untreated or pretreated with 50 µM AG490 for 4 h then treated with MSM for 24 h. (D) The relative levels of IGF-1R and GHR mRNA were determined using densitometric analysis and normalized to the amount of 18S. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p

Effects of methylsulfonylmethane (MSM) on osteoblast differentiation in primary murine bone marrow mesenchymal stem cells (MSCs).

<p>(A) Comparison of alkaline phosphatase (ALP) activity of various MSM treatments with varying culture durations. Cells were treated with various concentrations (0, 5, 10 and 20 mM) of MSM for 3, 5, and 7 days. ALP activity was measured by the release of p-nitrophenol from p-nitrophenyl phosphate and was normalized to the protein level. Osteoblastic mineralization was determined by Alizarin Red S staining (B) and von Kossa staining (C) at day 21. Cells were cultured in non osteogenic medium (NO) or osteogenic medium (OM) with various concentrations (0 and 20 mM) of MSM for 21 days (scale bar: 50 µm). Mineral nodules (more than 1 mm in diameter) were counted and expressed in histogram. Data shown are representative of three independent experiments. Asterisks indicate a statistically significant increase by ANOVA (***p<0.001).</p