| BackgroundApoptosis consists of two major signal pathways:the extrinsic death receptor pathway and the intrinsic mitochondrial pathway. Death receptor5(DR5) is a protein that belongs to tumor necrosis factor receptor (TNFR) superfamily. It contains a cytoplasmic death domain (DD) which can recruit Fas-Associated Death Domain (FADD) and pro-caspase8/10to form the Death-Inducing Signal Complex (DISC) when the receptor is trimerized. Subsequently, initiator caspase proteins are activated to lead the cleavage of downstream effectors. Development of pro-apoptotic agonists has been focused on DR5because of its target selectivity for malignant over normal cells. The activation of caspase8and downstream effectors can be inhibited by anti-apoptosis protein c-FLIP which prevents recruitment of pro-caspase8to DISC.The imbalance among the Bcl-2family members which have been defined as either anti-apoptotic or pro-apoptotic is essential for the modulation of intrinsic apoptosis signal pathway. The BH3-only protein NOXA is a p53transcriptional target in response to DNA damage. It has been reported to be involved in chemotherapeutic agent-induced apoptosis. NOXA can interact with pro-survival Bcl-2family protein Mcl-1, results in displacing Bax from the Mcl-1/Bax complex and freeing Bax to trigger the intrinsic pathway. This combination between NOXA and Mcl-1also promote proteasomal degradation of Mcl-1to enhance the mitochondrial apoptosis.Many chemotherapy drugs have been reported to induce ER stress response in cancer cells. ER stress is usually caused by accumulation of misfolded or unfolded proteins in the ER lumen. Three signal pathways are involved in ER stress. The transducers protein kinases IRE1and PERK and the transcription factor ATF6regulate the ER stress through their respective signaling cascades. When those misfolded or unfolded proteins are not resolved, ER stress is prolonged and then induces apoptosis. There are several mechanisms linking ER stress to apoptosis such as cleavage and activation of pro-caspase12, activation of ASK1and so on. Many studies have focused on the ER stress effector CHOP, which is a downstream target of ATF4. CHOP is a bZIP-containing transcription factor that can target several apoptotic genes including DR5and NOXA. The molecular mechanisms of ER stress-induced apoptosis is remain unclear.Cancer stem cells have many similar aspects with stem cells. Those cells express typical markers of stem cells and have the ability of self-renewal and differentiation. Cancer stem cells are also considered to be the origin of cancer cells and have more resistance to anti-cancer drugs. Many reports have indicated that cancer stem cells are correlated with poor prognosis. So, targeting cancer stem cell may be a promising strategy for cancer therapy.Parthenolide is a sesquiterpene lactone derived from the plant feverfew. It has been used to treat inflammation due to its ability of inhibiting NF-κB activity. Parthenolide has also been reported to play other roles such as promoting cellular differentiation, causing cell cycle arrest and inducing apoptosis. Its pro-apoptotic effect on cancer cells is known to trigger the intrinsic apoptotic pathway by elevating levels of intracellular reactive oxygen species (ROS) and alterating of Bcl-2family proteins. What’s more, recent studies have revealed that PTL could selectively eradicate acute myelogenous leukemia stem and progenitor cells. It is also demonstrated that PTL could preferentially inhibit breast cancer stem-like cells, but the molecular mechanism is still unclear.The transfer among components of cell plasma membrane system is often executed by vesicular transport. Vesicular transport is a highly organized orientational process. With energy provided by the hydrolysis of ATP, vesicular transport makes coated pits transport to the target organelle via the microtubules.Vesicular transport includes exocytosis and endocytosis. Based on the difference of coat proteins, endocytosis can be divided into clathrin-dependent endocytosis and clathrin-independent endocytosis. Receptor-mediated endocytosis is a program by which cells ingest extracellular proteins and other compounds depended on specific receptors on the cell surface. After endocytosis, receptors can be recycled back to the plasma membrane, or transferred from the late-endosome to lysosome for degradation. Endocytosis regulates multiple signaling pathways and is important to modulate cell polarization, movement, signal transmission, and growth.Many studies considered DR5to be the pivotal death receptor in a variety of cells. DR5is also considered to be the preferred target to exploit antibodies for cancer therapy. The localization appropriacy on plasma membrane is necessary for DR5to transfer ligand signals. Studies have found that some cancer cells without surface DR5such as breast cancer cells have stronger resistance to antibodies of TRAIL and its receptors.TRAIL and its receptor DR5can be rapidly internalized after TRAIL treated. The internalization can go through both the clathrin-dependent and the clathrin-independent endocytosis. It has been reported that activation of death receptor results in cleavage of several proteins in clathrin-dependent endocytosis by a caspase-denpendent machine. Inhibition of endocytosis can enhance the activation of caspase, and enlarge cell death furthermore.However, there are also data showed that in some cells, the total protein level of clathrin and adapter protein AP2is not directly related to the surface expression of DR4and DR5on plasma membrane. Other components of clathrin-dependent endocytosis may also participate in the process by which receptors are deficiency on plasma membrane. More research about endocytosis and related molecules involved in the lack of surface TRAIL receptor is required. Those research are important for the proper selection and design of anti-cancer drug.Numb is originally defined as an important molecule that decideed cell fate in Drosophila. Numb has a variety of abilities, including the decision of cell fate, regulation of asymmetric cell division, endocytosis, cell adhesion, cell migration, specific ubiquitin of substrates and so on.Numb can act as an endocytic protein, it exists on endosome, clathrin pits and vesicles. Numb is co-located with internalized cargos, and transfer from middle vesicle to endosome through all processes of endocytosis. Numb can be recruited to endocytosis organelles by combining with Eps15, but Numb and Eps15cannot be located in a same vesicle, indicating that Numb may be combined with other components of endocytosis.Methods1. The culture of tumor cells;2. SRB and MTT assays were executed to detect the survival of tumor cells;3. Flow cytometry assay were carried out to detect the cell cycle;4. The expression level of relevant proteins in cells were detected by western blot asssays;5. Apoptosis was detected by flow cytometry assay;6. RNAi technology was used to inhibit the expression of related genes;7. Gene cloning;8. Surface expression of DR5was detected by flow cytometry assay;9. Over-expression of genes was used to assay the effects on related proteins;10. Immunofluorescence was used to test surface expression of DR5;11. Immunoprecipitation was carried out to examine the interactions between proteins.Results1. PTL can obviously inhibit the cell survival of non-small cell lung cancer cells such as Calu-1,H1792A549, H1299, H157and H460, this inhibition was in a concentration dependent manner;2. PTL induced G0/G1cell cycle arrest in A549cells in a concentration dependent manner while induced G2/M cell cycle arrest in H1792cells;3. In A549, Calu-1, H1299and H1792cells, PTL induced the activation of pro-apoptosis protein caspases in a concentration and time dependent manner;4. PTL up-regulated the expression of death receptor5in A549, Calu-1, H1299and H1792cells in a concentration and time dependent manner, knocking-down of DR5significantly inhibited the caspase cascade activation and cell apoptosis induced by PTL;5. PTL inhibited the expression of anti-apoptosis protein c-FLIP in A549, Calu-1, H1299, H1792and H157cells, this inhibition is in a concentration and time dependent manner, over-expressing of c-FLIP could significantly inhibit the caspase cascade activation and apoptosis induced by PTL;6. PTL up-regulated the expression of pro-apoptosis protein NOXA in A549, Calu-1, H1299and H1792cells in a concentration and time dependent manner, while the expression of anti-apoptosis protein Mcl-1was inhibited in an opposite manner, knocking-down of NOXA up-regulated the expression of Mcl-1and inhibited the caspase cascade activation and cell apoptosis induced by PTL7. PTL up-regulated the expression of ATF4and CHOP, which are effector proteins endoplasmic reticulum stress pathway. This up-regulation were in a concentration and time dependent manner. Knocking-down of ATF4inhibited the expression of CHOP and PTL inducing caspase cascade actication, knocking-down of CHOP inhibited the expression of DR5and NOXA and the caspase cascade activation induced by PTL;8. PTL up-regulated the expression of endoplasmic reticulum stress pathway proteins such as IRE1α, Bip, p-eIF2a in a concentration and time dependent manner;9. Deletion of E-cad increases expression of stem cell marker Oct4and Sox2in A549cells;10. The same concentration of PTL had a stronger inhibitory effect on cell survival and caspase cascade activation in A549/shE-cad cells;11. The up-regulation of pro-apoptosis protein NOXA as well as down-regulation of anti-apoptosis protein c-FLIP and Mcl-1induced by PTL were enhanced in A549/shE-cad cells;12. The up-regulation of endoplasmic reticulum stress related proteins p-eIF2a, ATF4and CHOP induced by PTL were enhanced in A549/shE-cad cells, knocking-down of CHOP inhibits the up-regulation of NOXA and the activation of caspase3and PARP induced by PTL. 13. In A549, Calu-1, H1299and H1792cells, SAHA induced the activation of caspases in a concentration dependent manner;14. SAHA up-regulated the expression of death receptor5in A549and H1792cells in a concentration dependent manner, knocking-down of DR5had a stronger effect in the inhibition of the caspase cascade activation induced by SAHA;15. Anti-cancer drugs SAHA and PEM increased surface expression of DR5in A549and H1792cells;16. Bafilomycin A1and MG132could strengthen the up-regulated expression of DR5induced by PEM in A549cells, E-64d and MG132could strengthen the up-regulated expression of DR5induced by SAHA in both A549and H1792cells;17. In A549and H1792cells, SAHA and PEM inhibited the expression of Numb and NumbL in a concentration dependent manner;18. Knocking-down of Numb and NumbL enhanced the expression of DR5and caspase cascade activation induced by SAHA and PEM; while over-expression of Numb and NumbL increased the expression of DR5and caspase cascade activation induced by SAHA and PEM;19. Numb and NumbL inhibited surface expression of DR5;20. Numb and NumbL could interact with DR5.Conclusion1. PTL has significant anti-cancer effect, which is carried out by inducing apoptosis and cell cycle arrest;2. C-FLIP and DR5play an important role in the extrinsic apoptosis induced by PTL;3. PTL induces intrinsic apoptosis through NOXA-Mcl-1axis;4. PTL induces endoplasmic reticulum stress and then trigger apoptosis;5. PTL induces stronger endoplasmic reticulum stress and apoptosis in cancer stem like cells.6. An-cancer drugs SAHA and PEM induce apoptosis in non-small cell lung cancer cells by up-regulation of DR5; 7. SAHA and PEM increase the surface expression of DR5, inhibition degradation through lysosome pathway and proteasome pathway can enhance the DR5expression induced by anti-cancer drugs;8. Anti-cancer drugs up-regulate DR5and caspase cascade activation by inhibiting Numb and NumbL;9. Numb and NumbL inhibited surface expression of DR5;10. Numb and NumbL can interact with DR5.To sum up, our study found that PTL has the ability of anti-cancer by inducing apoptosis and cell cycle arrest, we propose the molecular mechanism by which PTL inducing apoptosis in non-small cell lung cancer cells:PTL activates the endoplasmic reticulum stress, increases the expression of ATF4and CHOP; CHOP serves as a transcription factor to increase expression of pro-apoptosis proteins DR5and NOXA, which were then cause extrinsic and intrinsic apoptosis. Anti-apoptosis protein c-FLIP participates in the PTL induced extrinsic apoptosis. We also found that PTL induces stronger endoplasmic reticulum stress and apoptosis in cancer stem-like cells, which provides a possible molecular mechanism to the selective effect of PTL towards cancer stem-like cells.Endocytic protein Numb and NumbL can combine with death receptors DR5. Anti-tumor drugs can increase the total and surface expression of DR5by inhibition of Numb and NumbL, then induce apoptosis in non-small cell lung cancer cells. Inhibition of lysosome and proteasome degradation pathway can enhance the DR5expression induced by anti-cancer drugs, Numb and NumbL reduce the surface expression of DR5probably by promoting endocytosis of DR5. Our study enriches understanding about endocytosis and related molecules involved in the lack of surface TRAIL receptor. |
