Loss of FADD and Caspases Affects the Response of T-Cell Leukemia Jurkat Cells to Anti-Cancer Drugs

Abstract

Programmed cell death (PCD) pathways play a crucial role in the response of cancer cells to treatment. Their dysregulation is one of the cancer hallmarks and one of the reasons of drug resistance. Here, we studied the significance of the individual members of PCD signaling pathways in response to treatment with common anti-cancer drugs using the T-cell leukemia Jurkat cells with single or double knockouts of necroptosis and/or apoptosis genes. We identified apoptosis as the primary cell death pathway upon anti-cancer drugs treatment. The cells with knocked out either Fas-associated protein with death domain (FADD) or all executioner caspases were resistant. This resistance could be partially overcome by induction of RIP1-dependent necroptosis through TNFR1 activation using combined treatment with TNF-α and smac mimetic (LCL161). RIP1 was essential for cellular response to TNF-α and smac mimetic, but dispensable for the response to anti-cancer drugs. Here, we demonstrated the significance of FADD and executioner caspases in carrying out programmed cell death upon anti-cancer drug treatments and the ability of combined treatment with TNF-α and smac mimetic to partially overcome drug resistance of FADD and/or CASP3/7/6-deficient cells via RIP1-dependent necroptosis. Thus, a combination of TNF-α and smac mimetic could be a suitable strategy for overcoming resistance to therapy in cells unable to trigger apoptosis.

Keywords: FADD; RIP1; RIP3; apoptosis; cancer; caspase; cell death; leukemia; necroptosis; ripoptosome.

Figure 1

Scheme of complexes formed in response to TNFα stimulation of tumor necrosis factor receptor 1(TNFR1). (A) Activation of TNFR1 by TNF-α leads to the trimerization of TNFR1 and to the formation of complex I containing TRADD, TRAF2, receptor-interacting serine-threonine kinase 1 (RIP1), cIAP1/2, and E3 ligases that mediate the ubiquitination of RIP1. Internalization of complex I, deubiquitination of RIP1, and recruitment of FAS-associated death domain protein (FADD) and caspase 8 form complex IIa, which may activate apoptosis through caspase 8. Active caspase 8 cleaves and inactivates RIP1 and necroptosis. (B) When caspase 8 is inactivated, complex IIb (ripoptosome), which includes RIP1, RIP3, mixed lineage kinase domain-like protein (MLKL), caspase 8, and FADD, is formed, and RIP1, RIP3, and MLKL are activated by phosphorylation and necroptosis is induced. →—activation, ┴—inhibition.

Figure 2

Cytotoxicity of anti-cancer drugs on knocked out cells. (A) Flow cytometry analysis of propidium iodide (PI)-stained Jurkat cells with knocked out one or more genes, whose products are involved in programmed cell death pathways showing percentage of dead cells after 48 h treatment with anti-cancer drugs camptothecin (Campt, 1 μM), etoposide (Eto, 5 μM), doxorubicin (Doxo, 0.15 μM), vinblastine (vinbl, 0.1 μM), Taxol (0.1 μM), and dinaciclib (Dina, 40 nM). When compared to wild type (WT) cells, statistically significant difference in drugs toxicity to knocked out cells was observed. Data are means ± S.E.M. of three independent experiments. * p < 0.01, ** p < 0.005 WT vs. knocked out cells. (B) Graph summarizing results of PI exclusion assays. The cells with disrupted genes for single caspases died more or equally to WT. Only caspase 3-deficient (CASP3) cells died less upon Campt and Doxo treatment. Cells with individually knocked out RIP1 or RIP3 died less than cells with knocked out MLKL or combination of apoptotic and non-apoptotic genes (RIP3/FADD, MLKL/FADD, and MLKL/CASP8).

Figure 3

Effect of TNF-α on Eto cytotoxicity. (A) Flow cytometry analysis of PI-stained cells showing percentage of the dead cells after 48 h treatment with Eto (5 μM) and TNF-α (10 ng/mL). TNF-α potentiated cytotoxicity of Eto in FADD- and CASP3/7-deficient cells. It had nearly no effect on CASP3/7/6-deficient cells. * p < 0.01. Eto vs. Eto/TNF-α. (B) Pretreatment with the pancaspase inhibitor (z-VAD, 20 μM) and/or RIP1 inhibitor necrostatin-1 (Nec-1, 20 μM) decreased dying of WT cells, while dying of CASP3/7/6- and FADD-deficient cells were affected only by Nec-1. Data are means ± S.E.M. of three independent experiments. Comparisons of Eto + TNF-α vs. inhibitors + Eto +TNF-α are shown.

Figure 4

Effect of smac mimetic (LCL161) and TNF-α. (A) Flow cytometry analysis of PI-stained cells showing percentage of the dead cells after 48 h treatment with LCL161 (10 μM) and TNF-α (10 ng/mL) and the effect of the pretreatment with the pancaspase inhibitor (z-VAD, 20 μM) and/or RIP1 inhibitor necrostatin-1 (Nec-1, 20 μM). LCL161 alone was nontoxic and TNF-α revealed a slight toxicity to WT cells and CASP3/7/6-, FADD-, and RIP3-deficient cells. The combination of these compounds significantly increased dying of the WT cells, CASP3/7/6, RIP3-deficient cells and also slightly of FADD-, RIP3/FADD-, and MLKL-deficient cells. RIP1-deficient cells almost were not affected by this treatment. z-VAD in combination with LCL161 and TNF-α did not affect CASP3/7/6- and FADD-deficient cells mortality, while Nec-1 effect was significant. Dying of WT cells and RIP3/FADD-, MLKL-, and RIP3-deficient cells was affected by both inhibitors. MLKL/FADD-deficient cells revealed weak sensitivity to treatment with LCL161/TNF-α, which was only slightly affected by z-VAD but not by Nec-1. Data are means ± S.E.M. of three independent experiments. * p < 0.01, ** p < 0.005 LCL161 vs. LCL161/TNF-α; LCL161 +TNF-α vs. inhibitors + LCL161/TNF-α. (B) Graph summarizing results of the effect of z-VAD and Nec-1 on combined treatment with LCL161/TNF-α.

Figure 5

Western blot analysis of markers of apoptosis (cleaved PARP; c-PARP) and necroptosis (pMLKL). (A) c-PARP detection after 3 h treatment of WT cells and FADD-, and CASP3/7/6-deficient cells with Eto (5 μM), Campt (1 μM), Taxol (0.1 μM), and LCL161/TNF-α (10 μM; 10 nM). In WT cells, c-PARP was present upon treatment with all compounds, while in FADD-deficient cells, basal level of c-PARP was present even in control cells. In CASP3/7/6-deficient cells, no c-PARP signal was detected. PCNA was used as a loading control. Total cell lysates were separated on 10% gels. Numeric values represent the ratio of band densities of c-PARP normalized to the corresponding PCNA and the control normalized to the corresponding PCNA. (B) Detection of MLKL and pMLKL necroptosis marker in WT cells and CASP3/7/6- and FADD-deficient cells after 24 h treatment with LCL161, TNF-α, and LCL161/TNF-α using Western blot analysis. Total cell lysates were separated on 10% gels. Numeric values represent the ratio of band densities of pMLKL to MLKL.

Figure 6

Electron microscopy of WT cells and CASP3/7/6- and FADD-deficient cells after 24 h treatment with Campt, Eto, and LCL161/ TNF-α (LCL)/TNF-a). In WT cells, there were only remains of dying and/or dead cells upon all treatments, showing necrotic morphology. Upon Campt (1 μM) treatment, cells were enlarged with lobate nuclei and contained autophagosomes (*) indicating induction of autophagy. Upon Eto (5 μM) and LCL161/TNF-α (10 μM; 10 nM) treatment, small number of mostly dead cells remained. On the contrary, CASP3/7/6- and FADD-deficient cells remained alive upon treatment with Campt and/or Eto and died only upon treatment with LCL161/TNF-α. Cells were enlarged and often contained multilobed nuclei. Mitochondria cristae disappeared upon Eto treatment. In the dead cells, we observed necrotic morphology. Bars 2 μm. In CASP3/7/6-deficient cells, Camp and Eto, and FADD-deficient cells, Campt and LCL161/TNF-α—bars = 5 μm.