Targeting Bcl-xL in esophageal squamous cancer to sensitize to chemotherapy plus TRAIL-induced apoptosis while normal epithelial cells are protected by blockade of caspase 9

TRAIL induces apoptotic cell death upon binding to either of two proapoptotic TRAIL receptors, TRAIL R1 (DR4) or TRAIL R2 (KILLER/DR5). Activation of the proapoptotic death receptors by TRAIL engagement induces the formation of a death-inducing signaling complex (DISC), which consists of receptor, FADD, as an adaptor, and caspase 8 as an initiator caspase. Once the DISC is formed, the caspase 8 is autoprocessed and activated by induced proximity. In FAS/ FASL-mediated apoptosis, two types of apoptotic signaling pathways exist, namely type I and type II. In type I cells, the activated caspase 8 could directly activate executioner caspase 3 without the involvement of the mitochondrial pathway. Even though either Bcl-2 or Bcl-xL may be overexpressed, the apoptotic signaling is not blocked in type I cells. In type II cells, there is little caspase 8 recruitment into the DISC; not enough to efficiently activate caspase 3, but enough to activate the mitochondrial pathway. Bid plays an important role in bridging signals from death receptors (extrinsic pathway) to the mitochondria (intrinsic pathway). Activated caspase 8 cleaves Bid and then, truncated Bid (tBid) translocates into mitochondria followed by Bax translocation, BAK oligomerization, cytochrome c release, and activation of caspase 9 and the other downstream caspases. The overexpression of either Bcl-2 or Bcl-xL could completely block the apoptotic signaling in type II cells. The observations that Bid is cleaved in TRAIL-mediated apoptosis, that some Bcl-2or Bcl-xL-overexpressing cells are still sensitive to TRAIL, and that in the presence of z-LEHD-FMK, a caspase 9 inhibitor, caspase 3 is activated in some cancer cells but not in other cancer cells suggest that the same types of pathways (type I and type II) may be utilized in TRAILmediated apoptosis. TRAIL is a promising agent for development as a cancer therapeutic because it appears to specifically kill transformed and cancer cells, whereas most normal cells appear to be resistant to TRAIL. Systemic administration of TRAIL is safe in mice and can kill breast or colon-xenografted tumors and prolong survival. However, the potential utility and safety of systemic administration of TRAIL has been questioned recently because of results showing sensitivity of human but not monkey or mouse hepatocytes to recombinant human TRAIL in vitro. Recently, we proposed the use of a specific caspase 9 inhibitor to circumvent that side effect based on the fact that hepatocytes are type II cells, and the inhibitor could rescue TRAIL hepatocytotoxicity without affecting the TRAIL killing effect towards cancer cells, many of which use a type I pathway in apoptotic signaling. Although TRAIL could specifically kill cancer cells, there are still many cancer cells that are TRAIL resistant. A number of mechanisms of TRAIL resistance have been described. For example, low or undetectable expression of DR4 or overexpression of TRAIL-decoy receptors, such as TRID (DcR1 or TRAIL-R3) or TRUNDD (DcR2, or TRAIL-R4), could block TRAIL-mediated apoptotic signaling. Loss of caspase 8 expression or high expression of c-FLIP make cancer cells also resistant to TRAIL. At the mitochondrial level, high expression of antiapoptotic Bcl-2 family proteins such as Bcl-2 or Bcl-xL might block death signal propagation if the cells use a type II pathway. Loss of expression of Apaf-1 documented in malignant melanoma could also provide a possible mechanism for TRAIL resistance at a postmitochondrial level. It was additionally reported that elevated AKT activity conferred TRAIL resistance on LnCap, a prostate cancer cell line, in a tissue-specific manner. Little is known about the effects of TRAIL on the human gastrointestinal epithelium. To gain a better understanding of TRAIL-mediated apoptosis, we chose for the present study human esophageal tissue, including eight squamous esophageal cancer cell lines and one normal primary esophageal epithelial cell. To assess TRAIL cytotoxicity towards squamous esophageal cancer cells, we performed the activecaspase 3 assay. With this assay, when more than 15% of cells revealed active-caspase 3 in a cell line, we defined the cell line as TRAIL sensitive. With this definition, three esophageal cancer cell lines (TE2, TE9, and TE12) were sensitive to TRAIL (Figure 1a). Cycloheximide reversed the TRAIL resistance in all cases. Previously, we reported that the expression levels of DR4 or c-FLIP were well correlated with TRAIL sensitivity. To evaluate determinants for TRAIL sensitivity in esophageal cancer cell lines, we performed Western blot immunostaining for expression of proteins involved in mediating the death signal (Figure 1b). Proapoptotic death receptors were highly expressed in TRAILresistant cancer cells and antiapoptotic decoy receptors were expressed in every cancer cell line. Furthermore, TRID expression in EPC2 was lower than in the cancer cell lines. Cell Death and Differentiation (2004), 1–5 & 2004 Nature Publishing Group All rights reserved 1350-9047/04 $25.00