Fig. 7. Antitumor effect and schematic diagram of cancer cell death by TGF-β downregulation.
a BALB/c athymic nude mice were injected with 8 × 106 A375 or HPAC cells in 100 μL. When the tumors reached an average size of 60–80 mm3, the nude mice received intratumoral injections of 1 × 109 plaque-forming units (pfu) of various kinds of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) in 50 μL of PBS or PBS alone on days 1, 3, and 5. Tumor volume was monitored and recorded every 2 days until the end of the study. Values represent the mean ± SE (five animals per group) (top). Asterisks indicate a significant difference compared to each given control (*p < 0.05; **p < 0.01). Overall survival was determined throughout a 31-day time course (bottom). b Representative immunohistochemical analysis of recombinant adenovirus-infected tumor sections was performed as follows. Three animals per group of BALB/c athymic nude mice were injected with 8 × 106 A375 or HPAC cells/100 μL and treated with intratumoral injections of 1 × 109 pfu/50 μL of various types of adenovirus (Ad-NC, Ad-shTGF-β1, or Ad-shTGF-β2) on days 1, 3, and 5. Tumors were collected on day 11 for histological analysis. Paraffin sections of tumor tissue were stained using anti-Hexon, anti-TGF-β1, and anti-TGF-β2 antibodies. c A TUNEL assay was performed on tissue sections to quantify apoptotic cell death, as described in the “Materials and methods” section. The percentage of TUNEL-positive cells was determined by counting the TUNEL-positive cells under 10 noncontinuous low-power fields. d TGF-β1 or -β2 downregulation can cause both NOX4-mediated ROS production and a reduction in Smad complexes (phospho-Smad2, 3 with Smad4) that translocate to the nucleus to bind to gene promoters for the expression of Trx/GSTM1. ROS triggered by decreased Akt activity can dissociate Trx or GSTM1 from ASK1–Trx and ASK1-GSTM1 complexes. ASK1 activation is also related to the reduction in Trx and GSTM1 gene expression, which results from decreased transcriptional activity of the Smad complex. In these ways, ASK1 was fully activated and induced cytotoxic tumor cell death via p38/JNK activation and induction of ER stress, which stimulated the escalation of ASK1 activation by completion of a sustained positive feedback loop circuit. Circled numbers indicate the chronological order of the signaling events leading to escalated cancer cell death