Molecular structure of DM-1.

<p>Molecular structure of DM-1.</p

Involvement of apoptotic members in melanoma tissues samples of B16F10 melanoma-bearing mice.

<p>Protein total extracts were used to analyzed Bcl-2, Bax, caspase-8, caspase-9 and caspase-3 after DTIC, DTIC+DM-1 or DM-1 treatment at the endpoint of the experiment. α-Tubulin expression was used as a loading control.</p

Liver (A); lungs (B); kidneys (C) and spleen (D) weight of untreated normal mice and B16F10 melanoma-bearing mice after DTIC, DM-1 or DTIC+DM-1 treatment.

<p>The values are expressed as mean ± s.d. Significance is indicated by #p<0.05 compared to untreated normal mice.</p

Macroscopic aspect of B16F10 melanoma-bearing mice on the 7^th, 10^th and 14^th days of treatment with DTIC, DM-1 or DTIC+DM-1 in comparison to control group.

<p>The measurements were made using a caliper rule.</p

Total weight and tumor mass of B16F10 melanoma-bearing mice on the 1st day of DTIC, DM-1 or DTIC+DM-1 treatment in comparison to the control group (A); Total weight and tumor mass of B16F10 melanoma-bearing mice on the 7^th day of DTIC, DM-1 or DTIC+DM-1 treatment in comparison to the control group(B); Total weight and tumor mass of B16F10 melanoma-bearing mice on the 14^th day of DTIC, DM-1 or DTIC+DM-1 treatment in comparison to the control group (C).

<p>The values are expressed as mean ± s.d. Significance is indicated by *p<0.05, **p<0.01 and ***p<0.001 compared to control.</p

Red blood cells (RBC) (A); reticulocytes (B); white blood cells (WBC) (C) and platelets (D) in peripheral blood of untreated normal mice and B16F10 melanoma-bearing mice after DTIC, DM-1 or DTIC+DM-1 treatment.

<p>The peripheral blood was collected and the cells were counted on the 1st, 7th and 14th days of treatment. The values are expressed as mean ± s.d. Significance is indicated by: *p<0.05, **p<0.01 and ***p<0.001 compared to control; and #p<0.05, ##p<0.01 and ###p<0.001 compared to untreated normal mice.</p

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

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

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

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