1. Galectin-9 induces apoptosis through the calcium-calpain-caspase-1 pathway
Masako Seki, Nozomu Nishi, Takanori Nakamura, Kazuhiro Nakamura, Mohammad J Abedin, Mitsuomi Hirashima, Yumiko Kashio, Naoko Yoshida J Immunol . 2003 Apr 1;170(7):3631-6. doi: 10.4049/jimmunol.170.7.3631.
Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.
2. Sequential caspase-2 and caspase-8 activation is essential for saikosaponin a-induced apoptosis of human colon carcinoma cell lines
Sung Hee Hong, Byeong Mo Kim Apoptosis . 2011 Feb;16(2):184-97. doi: 10.1007/s10495-010-0557-x.
In this study, we investigated the signaling pathways implicated in SSa-induced apoptosis of human colon carcinoma (HCC) cell lines. SSa-induced apoptosis of HCC cells was associated with proteolytic activation of caspase-9, caspase-3, and PARP cleavages and decreased levels of IAP family members, such as XIAP and c-IAP-2, but not of survivin. The fluorescence intensity of DiOC6 was significantly reduced after SSa treatment. CsA significantly inhibited SSa-induced loss of mitochondrial transmembrane potential and moderately inhibited SSa-induced cell death. SSa treatment also enhanced the activities of caspase-2 and caspase-8, Bid cleavage, and the conformational activation of Bax. Additionally, SSa-induced apoptosis was inhibited by both the selective caspase-2 inhibitor z-VDVAD-fmk and the selective caspase-8 inhibitor z-IETD-fmk and also by si-RNAs against caspase-2 and caspase-8. The selective caspase-9 inhibitor, z-LEHD-fmk, also inhibited SSa-induced apoptosis, albeit to a lesser extent compared to z-VDVAD-fmk and z-IETD-fmk, indicating that both mitochondria-dependent and mitochondria-independent pathways are associated with SSa-induced apoptosis. Both z-VDVAD-fmk and z-IETD-fmk significantly attenuated the colony-inhibiting effect of SSa. Moreover, inhibition of caspase-2 activation by the pharmacological inhibitor z-VDVAD-fmk, or by knockdown of protein levels using a si-RNA, suppressed SSa-induced caspase-8 activation, Bid cleavage, and the conformational activation of Bax. Although caspase-8 is an initiator caspase like caspase-2, the inhibition of caspase-8 activation by knockdown using a si-RNA did not suppress SSa-induced caspase-2 activation. Altogether, our results suggest that sequential activation of caspase-2 and caspase-8 is a critical step in SSa-induced apoptosis.
3. The caspase 9 inhibitor Z-LEHD-FMK protects human liver cells while permitting death of cancer cells exposed to tumor necrosis factor-related apoptosis-inducing ligand
N Ozoren, A D Moscioni, W S El-Deiry, K Kim, D T Dicker, T F Burns Cancer Res . 2000 Nov 15;60(22):6259-65.
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.
