Caspase-9 Inhibitor Z-LEHD-FMK

Object: Apoptosis is considered one of the most significant mechanisms in the pathogenesis of neuronal damage after spinal cord injury (SCI). This form of cell death occurs via mediators known as caspases. The aim of this study was to evaluate the neuroprotective effect of the caspase-9 inhibitor, z-LEHD-fmk, in a rat model of spinal cord trauma. Methods: Fifty-four Wistar albino rats were studied in the following three groups of 18 animals each: sham-operated controls (Group 1); trauma-only controls (Group 2); and trauma combined with z-LEHD-fmk-treated animals (0.8 microM/kg; Group 3). Spinal cord injury was produced at the thoracic level by using the weight-drop technique. Responses to SCI and the efficacy of z-LEHD-fmk treatment were determined on the basis of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining and light and electron microscopy findings in cord tissue at 24 hours and 7 days posttrauma. Six rats from each group were also assessed for functional recovery at 3 and 7 days after SCI. This was conducted using the inclined-plane technique and a modified version of the Tarlov motor grading scale. At 24 hours postinjury, light microscopic examination of Group 2 tissue samples showed hemorrhage, edema, necrosis, polymorphonuclear leukocyte infiltration, and vascular thrombi. Those obtained in Group 3 rats at this stage showed similar features. At 24 hours postinjury, the mean apoptotic cell count in Group 2 was significantly higher than that in Group 3 (90.25 +/- 2.6 and 50.5 +/- 1.9, respectively; p < 0.05). At 7 days postinjury, the corresponding mean apoptotic cell counts were 49 +/- 2.1 and 17.7 +/- 2.6, also a significant difference (p < 0.05). Electron microscopy findings confirmed the occurrence of programmed cell death in different cell types in the spinal cord and showed that z-LEHD-fmk treatment protected neurons, glia, myelin, axons, and intracellular organelles. Conclusions: Examination of the findings in this rat model of SCI revealed that apoptosis occurs not only in neurons and astrocytes but also in oligodendrocytes and microglia. Furthermore, immediate treatment with the caspase-9 inhibitor z-LEHD-fmk blocked apoptosis effectively and was associated with better functional outcome. More in-depth research of the role of programmed cell death in spinal cord trauma and further study of the ways in which caspases are involved in this process may lead to new strategies for treating SCI.

The present investigation was done to study the effect of caspase-9 inhibitor Z-LEHD-FMK, on in vitro produced buffalo embryos. Z-LEHD-FMK is a cell-permeable, competitive and irreversible inhibitor of enzyme caspase-9, which helps in cell survival. Buffalo ovaries were collected from slaughterhouse and the oocytes were subjected to in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC). The culture medium was supplemented with Z-LEHD-FMK at different concentrations i.e. 0 μM (control), 10 μM, 20 μM, 30 μM and 50 μM during IVM and IVC respectively. After day-2 post-insemination, the cleavage rate was significantly higher (74.20 ± 5.87% at P<0.05) in the group treated with 20 μM of Z-LEHD-FMK than at any other concentration. Same trend was observed in the blastocyst production rate which was higher at 20 μM (27.42 ± 2.94% at P<0.05). The blastocysts obtained at day-8 of the culture at different concentrations were subjected to TUNEL assay, to determine the level of apoptosis during the culture medium supplied with 20 μM Z-LEHD-FMK which showed apoptotic index significantly lower (1.88 ± 0.87 at P<0.05). There was a non-significant increase in total cell number in all Z-LEHD-FMK treated blastocysts. The quantitative gene expression of CHOP and HSP10 genes showed significant increase (P<0.05) in the group treated with 50 μM Z-LEHD-FMK, while, HSP40 showed significant increase (P<0.05) at 30 μM and 50 μM Z-LEHD-FMK concentrations. From the afore mentioned results we conclude that, Z-LEHD-FMK at 20 μM increased the cleavage and blastocyst rate of buffalo pre-implantation embryos also affecting the rate of apoptosis and cellular stress at various concentrations.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis of transformed and cancer cells but not of most normal cells. Recent studies have revealed an unforeseen toxicity of TRAIL toward normal human hepatocytes, thereby bringing into question the safety of systemic administration of TRAIL in humans with cancer. We found that SW480 colon adenocarcinoma, or H460 non-small cell lung cancer cell lines, which are sensitive to TRAIL, were not protected by the caspase 9 inhibitor Z-LEHD-FMK from TRAIL-induced apoptosis. However, a human colon cancer cell line HCT116 and a human embryonic kidney cell line 293, which are sensitive to TRAIL, were protected by Z-LEHD-FMK from TRAIL-mediated death. Both HCT116 and SW480 cells were protected from TRAIL by the caspase 8 inhibitor Z-IETD-FMK, dominant-negative FADD and cellular FLIP-s and interestingly both cell lines displayed caspase 9 cleavage to a similar extent after TRAIL exposure. We confirmed that normal human liver cells are sensitive to TRAIL. Moreover, we found that normal human liver cells could be protected from TRAIL-induced apoptosis by simultaneous exposure to Z-LEHD-FMK. A similar brief exposure to TRAIL plus Z-LEHD-FMK inhibited colony growth of SW480 but not HCT116 cells. Because some cancer cell lines are not protected from TRAIL-mediated killing by Z-LEHD-FMK, we believe that a brief period of caspase 9 inhibition during TRAIL administration may widen the therapeutic window and allow cancer cell killing while protecting normal liver cells. This strategy could be further developed in the effort to advance TRAIL into clinical trials.