| Caspase-dependent apoptosis is a typical mechanism by which most chemotherapeutic agents exert their antitumor activity. However, chemotherapy resistance of recurrent tumors that are common in patients is directly linked to the limiting success of cytotoxic drugs against cancers. Compelling evidence has demonstrated that chemotherapy resistance can also be attributed to acquisition of anti-apoptotic mechanisioms, because cancer cells often develop defects in apoptosis that provides a survival advantage and contributes to chemotherapy resistance.Therefore, therapies based on induction of non-apoptotic cell deaths, such as paraptosis, autophagic cell death, necroptosis, provide an effective strategy for treatment of malignant cancers that are multi-drug resistant and often associated with resistance to apoptosis.Paraptosis, oneform of non-apoptotic cell death characterized by extensive cytoplasmic vacuolation resulted from swelling of the ER and/or mitochondria, is caspase independent lacking morphological hallmarks of apoptosis.Though the mechanisms involved in triggering dilation of the ER and/or mitochondria are poorly defined, de novo protein synthesis and ER stressare believed to be essential for paraptotic process because protein synthesis inhibition attenuates the vacuolation.Enforcing the unfolded protein response (UPR), which can be initiated by ER stress also contribute to cell death through the three ER sensors: protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1α(IRE1α) or activating transcription factor 6 (ATF6; both a and β isoforms). Considerable efforts have been made to identifyER stress/UPR activavitors that specifically trigger these alternate cell death mechanisms as novel cancer treatment options.When autopsy cell death occurs, the cell cytoplasm produces a large number of free membrane structure, which makes the cell appear "bubble", which can be called the pre Preautophagosome. Tthe autophagy vacuolation further into the expansion, the formation of a lipid bilayer structure of the vacuolation, the vacuolation inside the cell is generally degeneration and necrosis of the cell and even some of the cell plasma, known as the autophagy vacuolation (Autophagosome). The origin of autophagic vacuoles is not clear, the general view that the source to the rough endoplasmic reticulum, or source in the late Golgi and membrane vesicles bulbs also suggested that the membrane structure is re synthesis. In mammals, the formation of autophagic vesicles process mainly consists of members of the Atg protein family and LC3 (Microtubule-associated protein 1 light chain 3, MAP1-LC3). Research shows that the Atg 12 may involved in autophagy (Preautophagosome) formation, and LC3 modification palys an important role of autophagy (Autophagosome) formation.Results:Part I The screening of naturally-occuring compounds that could induce tumor cell death1.Benzo(a)quinolizidine analogs induce cancer cell death through paraptosis and cytoplasmic vacuolationIn this thesis, we used human breast cancer cells (MDA-MB-231, MCF-7), human cervical carcinoma cell (Hela), human chronic myelogenous leukemia cells (K562), human prostate cancer cells (PC3, DU145) and human normal prostate epithelial cells (RWPE1) for the screening model, and the effect of these benzo(a)quinolizidine derivativeson cell proliferation was investigated in a panel of human cancer cell lines by MTT assay.The results showed that benzo(a)quinolizidine analogs could induce cytoplasmic vacuolation and cancer cell death in a low concentration.2.Marchantin C Synergistic with MG132 inducingcancer cell death and cytoplasmic vacuolationThe subject that I involved before have indicated that one of the bisbibenzylsderivatives, Marchantin C(MC), exertanti-tumor activity in vitro and in vivoin a manner of paraptosis. In this thesis, we further investigate the combination between MC and MG132, and detectedthe synergistic effectof the two compounds that inducingcancer cell death and cytoplasmic vacuolation.PartⅡ The mechanism of 22b inducing cancer cell drath and cytoplasmic vacuolationPrevious anti-proliferation screening of benzo(a)quinolizidine analogs indicated that these alkaloids could provide two kinds of cell death passway, apoptosis and paraptosis. Thusprovide an effective strategy for treatment of malignant cancers that are multi-drug resistant and often associated with resistance to apoptosis.Among thebenzo(a)quinolizidine analogs, we chosed 22b as potential compound to find out the mechanism of nonapoptosis-like cell drath and cytoplasmic vacuolation in prostate cancer.1.22b could inducescell cycle arrest, cytoplasmic vacuolation and apoptosis.Given the inhibitory effect of compound 22b on PCa cells, we then analyzed changes in cell cycle distribution and apoptosis in response to 22b.According to the experiment result,22b induced cell cycle arrest at G0/G1 phase, and induce apoptosis in a concentration depend manner. However, pan-caspase inhibitor Z-VAD-fmkpartially rescued 22b-induced cell death, indicating that caspase-independent mechanisms also contributed partially to the 22b-induced cell death.2.22b-mediated paraptosis depends on ER stress and protein synthesis.(1)22b can induce ER stress significantly.Compound 22b make the sign of ER stress proteinGRP78 continuely increase, and the expression of p-eIF2alpha also presents the same trend while the total protein eIF2 alpha did not change, the aboveresult evidenced that 22b can indeed induce ER stress.(2)22b-Mediated Paraptosis Depends on ER Stress and Protein Synthesis. To investigate the requirement of ER stress in the formation of vacuoles, we included 4-phenylbutyricacid (4-PBA), an ER stress inhibitor, to suppress 22b-mediated ER stress.The 22b-induced cell drath and vacuoles were well abolished in the presence of 4-PBA, the same result occurred while the exest of translation inhibitorcycloheximide (CHX), or transcriptional inhibitor actinomycin-D (Act-D), highlighting the importance of ER stress in regulation of vacuole formation which is closely correlated with22b-mediated cell death.(3)proteasome inhibitors that are able to trigger ER stress could facilitate 22b-induced vacuolation and cell death. CompuSyn software was used in analysis of effect of 22b combined with 39 (MG132) and Bortezomib, the proteasome inhibitors that are able to trigger ER stress. The result showed that the combination of 22b and MG132 yielded a strong synergistic effect. Similarly, Bortezomib also has the synergistic effects.So ER stress play an important role in22b-induced vacuolation and cell death.3.22b-induced cytoplasmic vacuolationrequires elevated LC3.(1)The role of autophagy in22b-induced vacuolation and cell death.A pH-sensitive LC3-reporter plasmid was transfected into cancer cells, and we found that 22b caused impaired autophagosome-lysosome fusion. The data that LC3B-Ⅱ and p62 levels did not reduce over time during treatments also indicates that autophagosomes may not be efficiently fused with the lysosomes.The persistent upregulated LC3-Ⅱ and p62, as well as the failed protective effect from 3-MA and E-64d, demonstrated that 22b-induced vacuolation was independent of classic antophagy and lysosomal degradation.(2)Thenon-autophagic role of LC3 in 22b-induced cytoplasmic vacuolation.depletion of LC3 by targeting-LC3 siRNA almost completely abolished 22b-induced extensive level of vacuoles. Consistent with the reports that the increased LC3-Ⅱ is not specific for autophagy but also plays a nonautophagic role that occurs during cytoplasmic vacuolation.Thus, the 22b-induced vacuolation associated with formation of LC3-Ⅱ-positive non-autophagic vacuoles contributes to its cytotoxicity.4.In Vivo Anticancer Activity of 22b.Together, our data in animal experimentclearly demonstrated that 22bevidently exhibited antitumor effect through elicitation of paraptosis-like cell death with less toxicity.ParⅢ The mechanism of synergistic effect between MC and MG132 inducing cancer cell drath and cytoplasmic vacuolation1.In Vivosynergistic effect between MC and MG132.The result showed that the combination of MC and MG132 yielded a strong synergistic effect in the animal expriment. Compare to the positive control group, the combination group have more antitumor effect as well as less toxicity. 2. PLIN2 may play an important role in the formation of vacuolation that caused by the synergistic effect between MC and MG132.(1)According to the results of gene chip, we screened out the increase with more than 5-fold differences in gene and further selected PLIN2 to sdudythe mechanism of synergistic effect between MC and MG132 inducing cancer cell drath and cytoplasmic vacuolation(2)We used specific PLIN2 siRNA to knock down PLIN2 protein levels respectively. The vacuolation that induce by the combination of MC and MG132 were significantly eliminated, suggesting PLIN2 may play an important role in the formation of vacuolation that caused by the synergistic effect between MC and MG132.Conclusion:1.We chose 22b and MC to investigate the mechanism of paraptosis in cancer cells, by screening of two types of natural compounds.2.22b could inducescell cycle arrest, cytoplasmic vacuolation and apoptosis.3.22b-mediated paraptosis depends on ER stress and protein synthesis.22b-induced vacuolation was independent of classic antophagy and lysosomal degradation, but requires the non-autophagic role of LC3.4.The resultin animal experimentdemonstrated that MC couldsynergistic with MG132,and evidently exhibited antitumor effect through paraptosis-like cell death with less toxicity.5.PLIN2 may play an important role in the formation of vacuolation that caused by the synergistic effect between MC and MG132.Novelty and defects:1.Novelty1.1 We first reported benzo(a)quinolizidine analogs could induce paraptosis and used 22b as novel model to investigate the interrelationship of ER stress, protein synthesis in 22b-induced paraptosis. And first time reported 22b-induced vacuolation depends on the non-autophagic role of LC3. However, how LC3 affect the formation of vacuoles needs to be further study.1.2 We first reported MC could synergistic with MG132,and evidently exhibited antitumor effect through paraptosis-like cell death with less toxicity. And first time reported PLIN2 and may play an important role in the formation of vacuolation that caused by the synergistic effect between MC and MG132. However, how PLIN2 and affect the formation of vacuoles needs to be further study.2.defects2.1 LC3 plays an important role in non-apoptotic cell death dependent on the autophagy pathway and non autophagic role, it is reported that ATF4 is increased significantly after the endoplasmic reticulum stress, and through the role of the transcription factor may regulate the expression of LC3. However, if there are other regulatory mechanisms still need further study. The next step will be to find the relationship between the formation of vacuoles and the formation of vacuoles by looking for the interaction proteins with LC3.2.2 previous results show that marchantin C can inhibit the proteasome activity. When combined with the proteasome inhibitor MG132. Marchantin C and MG132 has a synergistic effect. If marchantin C and MG132 on proteasome inhibition is a complementary role, or whether there are other mechanisms in addition to inhibition of the proteasome are still not clear. So the targets of marchantin C need further verification and discussion.2.3 PLIN2 is in relation to fat metabolism, but it is how to involved with the synergistic effect.of marchantin C and MG132 induced cytoplasmic vacuole formation is still not clear., The upstream regulation of PLIN2 expression still needs to be explored. This part of work is ongoing. |
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