Caspase-8 Induced Cancer Cell Death Via Disrupting The Homeostasis Of Lysosomes

ObjectivesIt has been well established that caspase-dependent apoptosis is a very important way of cell death,and it is also an effective pathway that various tumor cytotoxic drugs target.Caspase-8,a well-characterized initiator of apoptosis,has been reported to play a number of non-apoptotic roles in cells.Cell death is a complex process in which multiple organelles are involved.A lot of experimental evidence accumulated has unveiled that irreversibly damaged cells or potentially harmful cells were eliminated via intracellular exquisite regulation system.Lysosome,so named the digestion of the cargo,can contribute biomass materials for macromolecular synthesis.In addition,the effective lysosomal function can be regarded as energy source in rapidly dividing and invasive cancer cells.Accordingly,in the progression of cancer,there could be a lot of dramatic changes in lysosomal volume,cellular distribution,lysosome PH or integrity of lysosome membrane.Containing high levels of hydrolytic enzymes organelle,lysosomes,also collaboratively participate in the process of cell death.The mission of the study is to explore the non-apoptotic function of Caspase-8 in tumor cell,and seek a new mechanism of cell death by blocking the positive feedback cascade of apoptotic effects.Not only that,we will determine the exact mechanism,how ectopic expression of caspase-8 destroyed the lysosome to kill cancer cells,and explore the relationship with lysosomal homeostasis,lysosomal dysfunction.Finally,it provides a theoretical basis for caspase-8 derived short peptide to target lysosomal therapy.The project will contribute to the deeper appreciation of the role of caspase-8 in cancer,and provide a new strategy for cancer therapy.Methods1.The CRISPR/Cas9 technology was performed to knock out the caspase protein at the genetic level in tumor cells,and we evaluated validity of knockout by genome sequencing,western blot,and functional assay.This technology eliminated effects of small amounts of protein residues by RNA interference technique,and the model was used to definite the initiation and activation process of the apoptotic pathway in HeLa and MCF-7 cells.2.The hallmarks of classical apoptosis were detected by western blot in overexpression of caspase-8 cancer cells.Annexin V/PI flow cytometric studies and the GFP-based caspase-3 activity indicator(GC3AI)was utilized to monitoring the occurrence of apoptosis events.3.To verify the relationship between proteolytic enzyme function and cell death by reconstructing caspase-8 mutants or knockdown the protein at downstream of caspase-8.4.Detection whether protection of mitochondria could rescue the caspase-8 induced cancer cell death,by overexpressing Bcl-xL and Bcl-2 anti-apoptotic proteins.5.Based on HeLa cells,the stably knocking out caspase-3/7/8/9 was generated by gene knockout technique,called 4KO cell line.The proliferation ability of 4KO cells was analyzed by colony formation assay and cell growth assay.Wound healing assay and transwell migration assay were used to detect the effect of 4KO cell line on cell migration.6.In the model of 4KO,we analyzed the relationship between the blocked cascade amplification pathway and the exogenous expression of caspase-8 lethal phenomenon.7.The distribution and morphology of the lysosome and the lysosomal PH were investigated in expression of caspase-8 cancer cells,by LysoSensor,lysoTracker and acridine orange stain.8.Immunofluorescence and lysosomal LGALS puncta assay were further used to confirm lysosomal membrane permeabilization.9.Truncated caspase-8 plasmids were constructed.Co-immunoprecipitation was performed to analyze the candidate domain interacted with the V-ATPase complex on the lysosome member.Truncation of caspase-8 overexpression stable transfected cell line was established by lentiviral system.The sensitivity of GFP-P10 4KO cell to lysosomotropic drugs was tested by Cell viability assay.Results1.The apoptosis is dependent on the transmission of apoptotic signals by mitochondrial pathway in HeLa and MCF-7 cells.Overexpression of Bcl-xL and Bcl-2 to protect mitochondria or knock down Bid or Bax protein,which reduced MOMP,can block the apoptotic process.2.Caspase-8 overexpression cancer cells lead to a significant decreased in cell number.Western blot detected the hallmarks of classical apoptosis,including activated caspase-8,caspase-3 and caspase-7,and the poly(ADP-ribose)polymerase(PARP)cleavage in reintroduced caspase-8 cells.The observed fluorescence of GC3 AI and annexin V/PI flow cytometric studies demonstrated caspase-8 initiated cell death through activating the caspase dependent-apoptosis,regardless of formation of DISC.3.Overexpression of caspase-8 uncleavable(C8UC)or catalytic inactive mutation(C8CI)also leads to cell death.Even though protection of mitochondria,were insufficient to rescue cell death caused by caspase-8.4.A model of 4KO cell lines based on HeLa,knocked out all of caspase-3/7/8/9 at the gene level.It exhibited strong resistance to TNF-?,ABT737 and cisplatin induced apoptosis,but did not survive in the present of exogenous caspase-8.5.4KO cells and KOC8 cell that knocked out caspase-8 alone conferred increased cell proliferation ability and decreased the migration ability.6.The lysosomal function was impaired in the transfected caspase-8 cancer cells.Alkalizations of lysosomes were detected by LysoSensor Green and acridine orange staining.Moreover,the accumulation of LGALS-3 puncta and the dispersive distribution of cathepsin L indicated lysosomal destabilization.7.Our study also demonstrated that caspase-8 was physically associated with the subunit of vacuolar ATPase,inhibiting the activity of ATPase,thereby increased the pH in lysosome.This process was mainly through the interaction between the P10 small subunit and V-ATPase.8.P10 endowed the ability of interaction with ATPase,enhanced cell sensitivity to lysosomal stress.ConclusionIn this study,we revealed a specific pathway that caspase-8 induced cancer cell death beside the role of caspase-8 in apoptotic signaling cascade.Overexpression of caspase-8 uncleavable or catalytic inactive mutation lead to cell death,and p10 domain of caspase-8 is also capable of inducing cell death.Caspase-8 is physically associated with the subunit of V-ATPase to inhibit the activity of proton pump,resulting in increased lysosomal PH.Continued lysosomal alkalization promotes lysosomal selective or partial destabilization,triggering the irreversible cell death.Overexpression of P10 not only leads to cell death,but also enhances sensitivity to lysosomal drugs.Furthermore,the research will contribute to the deeper appreciation to the role of caspase-8 in cancer,and provide new potential targets for tumor therapy,especially for apoptosis resistance cancer.