Figure 6.

The in vivo effects of long-term rapamycin and doxycycline treatment in ZR75.1-derived xenografts. (a) 3-week-treated tumour xenografts were processed for transmission electron microscopy. Intact mitochondria were presented in untreated tumour sections, the number of autophagosomes was increased after Rapamune treatment; accompanied, and damaged mitochondria could be observed in doxycycline-treated samples. After administrating combined Rapamune and doxycycline treatment, collapsed mitochondria appeared in autophagic vacuoles. Mitochondria (M; clamped mitochondria were framed with white ellipsoids), autophagic vacuoles (arrow); nucleus (N); multivesicular bodies (MVB). (magnification: 20,000×). (b) Immunohistochemistry staining for analysing the expression of cleaved caspase-3 (apoptosis marker), LC3 (both LC3-B-I and II autophagosome proteins) and TOM20 (mitochondrial marker) in ZR75.1 xenografts. Treatments were administered for 21 days; after 21-day therapy, the drug withdrawal was also tested for an additional 21 days ($\frac{21 days R+D}{+21 days Ø}$). Images show representative immunostainings. DAB substrate was used for developing immunoreaction (brown), and specimens were counterstained with haematoxylin. Scale bar in the first photo—labels 50 µm. (Co—control; R—Rapamune 3 mg/kg; D—doxycycline 5 mg/kg; R+D—Rapamune + doxycycline combination; Ø no treatment).