Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

<div><p>Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and ⁷Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.</p> </div

Electron microscopy of melanoma cells from control, irradiated control, BNCT 1 and 7 days groups.

<p>(A,C) Detail of the preserved chromatin in the nuclei of melanoma cells from control and irradiated control groups. (B, D) Illustration of the high density population. By contrast, in panels (E, G, H), melanoma cells show a markedly condensed chromatin close to the nuclear membrane (arrows) 1 and 7 days after BNCT. (F) Decrease in cell density after BNCT. The organelles inside the melanoma cells appeared to be degenerated in both BNCT groups.</p

TUNEL, caspase 3- and caspase 8-stained sections and melanoma morphometric analysis of control, irradiated control, BNCT 1 day and 7 days groups.

<p>(A) In control and irradiated groups (×400), apoptotic melanoma cells, i.e., those stained with TUNEL, caspase 3 and 8 (arrow), were sparse. In BNCT 1 and 7 day groups (×400), many melanoma cells were in apoptosis. Graphic plots show an increase in apoptotic melanoma cells as determined by (B) TUNEL, (C) caspase 3 and (D) caspase 8 staining in BNCT 1 and 7 day groups. All results are expressed as mean ± s.d. ns: not significant compared to control. *p<0.05; **p<0.01; ***p<0.001 compared to control.</p

Hematoxylin and eosin-stained sections of malignant melanoma in control, irradiated control, BNCT 1 and BNCT 7 days groups.

<p>In the control and irradiated groups, malignant melanoma cells were preserved and composed of large cells with atypical nuclei and abundant cytoplasm. Normal mitosis (blue arrows) and aberrant mitosis (yellow arrows) were both observed. Necrosis was absent in both groups of melanoma. By contrast, extensive necrosis (nec), pycnotic nuclei (black arrows) and acidophilic cytoplasm (green arrows) were present in the malignant melanoma of BNCT 1 and BNCT 7 day groups. Furthermore, the BNCT groups also presented aberrant mitosis.</p

Expression of extrinsic apoptotic markers in B16F10 melanoma cells and normal melanocytes (mean ± s.d.) determined by flow cytometry.

<p>(A) Expression of TNF-R1 in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (B)Expression of TNF-R1 in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (C) Expression of cleaved caspase 8 in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (D)Expression of cleaved caspase 8 in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITC-conjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p<0.05; **p<0.01; ***p<0.001 compared to control.</p

Expression of Ki67 in B16F10 melanoma cells and normal melanocytes (mean ± s.d.) determined by flow cytometry.

<p>(A) Ki67 expression in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (B)Ki67 expression in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITC-conjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p<0.05; **p<0.01; ***p<0.001 compared to control.</p

Determination of collagen-related markers in B16F10 melanoma cells and normal melanocytes (mean ± s.d.).

<p>(A) Synthesis of soluble collagen in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (B) Synthesis of soluble collagen in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (C) Expression of ECM collagen in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (D)Expression of ECM collagen in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (E) Expression of Hsp47 in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (F)Expression of Hsp47in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITC-conjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p<0.05; **p<0.01; ***p<0.001 compared to control.</p

Expression of intrinsic apoptotic markers in B16F10 melanoma cells and normal melanocytes (mean ± s.d.) measured by flow cytometry.

<p>(A) Expression of Bad in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (B)Expression of Bad in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control).(C) Cytochrome c expression in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (D)Expression of cytochrome c in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITC-conjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p<0.05; **p<0.01; ***p<0.001 compared to control.</p

Melanoma cell death after BNCT.

<p>(A) Tumor volume of mice bearing B16F10 melanoma during twenty-one days. On the fourteenth day after B16F10 melanoma implantation, the BNCT group received BPA and was irradiated with thermal neutrons. The mice were sacrificed and analyzed 1 or 7 days after BNCT treatment. (B) Western blotting analyses of caspase 3, 7, 8, 9, Bcl-2 and Bax in melanoma tissue. (C, D) mRNA expression levels of caspase 3 and 8, respectively, quantified by RT-PCR.</p

Immuno-expression of IL-1β, IL-10, IL-17 and TNFα in the synovial sera from the IA, IA/Suppl and Suppl groups.

<p>Values are the means (±SEM) of 10 animals in each group. All values were completed in a few random, now coincident fields supplemented with daily doses for 30 days. Groups were compared using ANOVA. IA vs Suppl (p<0.01), IA vs IA/Suppl (p<0.01), Suppl vs IA (p<0.01).</p