So far, we tested cancer cell lines which are not expressed RIP3 and we wonder whether this sensitization is related with RIP3 expression. To rule out the dependency, we took the HT-29 cells which is well studied in RIP3-dependent MLKL-mediated necroptotic cell death (S. He et al., 2009). HT-29 cells were depleted MLKL and treated with different concentration of TRAIL.
Similar with cells lacking RIP3 expression, HT-29 cells were sensitized TRAIL-induced cell death (Figure 4A) and appeared fasten PARP / caspase 3 cleavage by MLKL depletion (Figure 4B) indicating RIP3-independent sensitization.It was further confirmed in RIP3 depleted HT-29 cells showing that RIP3 depleted HT-29 cells did not response to TRAIL-induced sensitization than HT-29 cells but still sensitive in MLKL depleted HT-29 cells indication that MLKL depletion may cause sensitization in TRAIL-induced cytotoxicity (Figure 5). Consistent with known RIP3 and MLKL function in necroptosis, these cells abolished TNF or TRAIL-induced RIP3-dependent necroptosis in both MLKL- or RIP3-depleted HT-29 cells (Figure 6A-B). We further examined MLKL dependency in necroptosis in response to chemotherapeutic agent, etoposide. Previous our report suggested that DNA damaging agents activate MLKL-dependent necroptosis and MLKL knockdown in HT-29 cells inhibited etoposide cytotoxicity (Koo et al., 2015).
Consistent with this, MLKL-depleted HT-29 cells inhibited etoposide cytotoxicity (Figure 6C) suggesting that MLKL promotes cell death through activation of necroptosis pathway. In HeLa cells, when RIP3 is ectopically expressed, depletion of MLKL is response to TRAIL sensitivity (Figure 7A).
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As expectedly, cells endogenously expressing MLKL acquired increased sensitivity to TRAIL when MLKL is knocked down by showing the PARP cleavage (Figure 7B). Taken together, these data suggested that depletion of MLKL enhances TRAIL-induced cell death in regardless of RIP3 status.
TRAIL binding to its death receptors DR4/DR5 induces the formation of DISC by the recruitment of FADD and 8 and then activated caspase-8 can further activate effector caspase-3 and -7, leading to cell-extrinsic apoptosis (Falschlehner, Emmerich, Gerlach, & Walczak, 2007). In the TRAIL-induced apoptosis signal, the functions of MLKL remain unclear. To verify the TRAIL-induced sensitization by MLKL depletion is typical apoptosis processes, we treated cells with pan caspase inhibitor zVAD which functions to inhibit apoptosis under TRAIL-induced cell death conditions.
As shown in Figure 8, various cancer cells showed lost their sensitivity to TRAIL by zVAD treatment in absent or presence of MLKL. Same as this, zVAD completely abolished TRAIL induced cell death and PARP cleavage in RIP3 expressing cancer cells (Figure 9A-B) indicating MLKL contributes to apoptosis, as evidenced by the following: (1) MLKL depleting increases TRAIL-upregulated c-PARP /c-caspase 3 level (2) MLKL depleting remarkably increases cell death (3) MLKL depletion-mediated sensitization of TRAIL-cytotoxicity is reduced by zVAD treatment.
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Figure 4. Depletion of MLKL accelerates TRAIL-induced death in RIP3-expressing cell line, HT-29.
(A) HT-29 cells expressing non-silencing or MLKL shRNA were analyzed by western blotting (left panel). These cells were dose dependent treated with TRAIL for 48 hours and cell viability was analyzed by MTT assay (right panel). The results are showed as means ± SEM. *P < 0.05, **P < 0.01, ***P
< 0.001.
(B) HT-29 cells stably expressing non-silencing or MLKL shRNA were treated with TRAIL in time dependent manner. The cells were harvested and lysates were analyzed by western blotting.
A
B
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Figure 5. TRAIL-induced cell death was sensitized by MLKL depletion, independent RIP3.
HT-29 cells expressing non-silencing, RIP3 shRNA or MLKL shRNA were analyzed by western blotting (left panel), and these cells were dose dependent treated with TRAIL for 48 hours. Cell viability was analyzed by MTT assay (right panel). The results are showed as means ± SEM. *P < 0.05, **P < 0.01,
***P < 0.001.
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Figure 6. MLKL promotes cell death through activation of necroptosis pathway.
(A, B) HT-29 cells stably expressing non-silencing, RIP3 shRNA, or MLKL shRNA were treated with TRAIL or TNF + SMAC mimetic + zVAD. Cell viability was analyzed by MTT assay.
(C) HT-29 cells stably expression non-silencing control or MLKL shRNA were treated etoposide for 48 hours. Cell viability was analyzed by MTT assay.
The results are showed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
A B
C
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Figure 7. Depletion of MLKL is response to TRAIL sensitivity, ectopically expressed RIP3.
(A) RIP3-expressing HeLa cells expressing non-silencing control or MLKL shRNA cells were dose dependent treated with TRAIL and cell viability was analyzed by MTT assay (upper panel) or phase-contrast microscopy (bottom panel). The results are showed as means ± SEM. *P < 0.05, **P < 0.01, ***P
< 0.001.
(B) RIP3-expressing HeLa cells expressing non-silencing control or MLKL shRNA were treated with TRAIL in dose dependent manner. The lysates were analyzed by western blotting.
A B
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Figure 8. TRAIL-induced sensitization by MLKL depletion is typical apoptosis processes.
HeLa, H2009 and HCC4006 cells expressing non-silencing control or MLKL shRNA were pretreated with zVAD for 1 hours and then treated with TRAIL.
Cell viability was analyzed by MTT assay. The results are showed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 9. TRAIL induced cell death and PARP cleavage were blocked by zVAD in RIP3 expressing cancer cells.
(A) HT-29 and RIP3-expressing HeLa cells expressing MLKL shRNA, or non-silencing control were pretreated with zVAD (20 μM) for 1 hours and then treated with TRAIL. Cell viability was analyzed by MTT assay.
(B) RIP3-expressing HeLa cells expressing MLKL shRNA, or non-silencing control were pretreated with zVAD for 1 hours and then treated with TRAIL (5 ng/mL). Cell lysates were analyzed by western blotting.
A
B
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