Liying Zhou

HP55-coated capsule containing PLGA/RS nanoparticles for oral delivery of insulin

In this work, we designed and developed a two-stage delivery system composed of enteric capsule and cationic nanoparticles for oral delivery of insulin. The enteric capsule was coated with pH-sensitive hydroxypropyl methylcellulose phthalate (HP55), which could selectively release insulin from nanoparticles in the intestinal tract, instead of stomach. The biodegradable poly(lactic-co-glycolic acid) (PLGA) was selected as the matrix for loading insulin. Eurdragit(®) RS (RS) was also introduced to the nanoparticles for enhancing the penetration of insulin across the mucosal surface in the intestine. The nanoparticles were prepared with the multiple emulsions solvent evaporation method via ultrasonic emulsification. The optimized nanoparticles have a mean size of 285nm, a positive zeta potential of 42mV. The encapsulation efficiency was up to 73.9%. In vitro results revealed that the initial burst release of insulin from nanoparticles was markedly reduced at pH 1.2, which mimics the stomach environment. In vivo effects of the capsule containing insulin PLGA/RS nanoparticles were also investigated in diabetic rat models. The oral delivered capsules induced a prolonged reduction in blood glucose levels. The pharmacological availability was found to be approximately 9.2%. All the results indicated that the integration of HP55-coated capsule with cationic nanoparticles may be a promising platform for oral delivery of insulin with high bioavailability.

Dynamics and structure of the Bax-Bak complex responsible for releasing mitochondrial proteins during apoptosis.

Bax and Bak are known to play a central role in facilitating the release of mitochondrial intermembrane proteins during apoptosis. The detailed mechanism, however, is still not clear. Using live cell imaging techniques, we showed here that Bax underwent four distinct stages of dynamic redistribution during UV-induced apoptosis. At stage I, Bax was distributed diffusely in the cytosol. About an hour after UV treatment at stage II, Bax started to translocate to mitochondria and distributed uniformly at the mitochondrial outer membrane (MOM). Within a few minutes, at stage III, Bax and Bak began to form small complexes at the MOM. Later, at stage IV, these Bax and Bak complexes expanded to become large clusters. We found that the formation of Bax-Bak small complexes at stage III was responsible for permeabilizing the MOM to release cytochrome c and Smac. Using a FRET technique, we further showed that Bax binds to Bak within the complex formed at the MOM during stage III. Finally, using a quantitative fluorescence measurement, we determined that the Bax-Bak complex was about 0.25 μm wide and composed of more than 100 protein molecules. These findings suggest that the Bax-Bak structure responsible for releasing mitochondrial proteins during apoptosis is not channel-like.

Smac/DIABLO and cytochrome c are released from mitochondria through a similar mechanism during UV-induced apoptosis.

During apoptosis, a key event is the release of Smac/DIABLO (an inhibitor of XIAP) and cytochrome c (Cyt-c, an activator of caspase-9) from mitochondria to cytosol. It was not clear, however, whether the releasing mechanisms of these two proteins are the same. Using a combination of single living-cell analysis and immunostaining techniques, we investigated the dynamic process of Smac and Cyt-c release during UV-induced apoptosis in HeLa cells. We found that YFP-labeled Smac and GFP-labeled Cyt-c were released from mitochondria in the same time window, which coincided with the mitochondrial membrane potential depolarization. Furthermore, using immunostaining, we found that the endogenous Smac and Cyt-c were always released together within an individual cell. Finally, when cells were pre-treated with caspase inhibitor (z-VAD-fmk) to block caspase activation, the process of Smac release, like that of Cyt-c, was not affected. This was true for both YFP-labeled Smac and endogenous Smac. These results suggest that in HeLa cells, both Smac and Cyt-c are released from mitochondria during UV-induced apoptosis through the same permeability transition mechanism, which we believe is triggered by the aggregation of Bax in the outer mitochondrial membrane to form lipid-protein complex.

A new anticancer compound, Oblongifolin C, inhibits tumor growth and promotes apoptosis in HeLa cells through bax activation

Oblongifolin C (OC) was identified as a potent apoptosis inducer from an herbal plant, Garcinia yunnanensis, during our previous bioassay‐guided drug screening. In this study, we investigated the signaling pathways through which OC activated apoptosis in HeLa cells. We also compared the IC50 values of OC with that of etoposide, paclitaxel and vinblastine in multiple cancer cell lines including HER2 and P‐glycoprotein overexpressing cells. In addition, the in vivo antitumor effect of OC was studied in nude mice model. Our results showed that OC induced a caspase‐dependent apoptosis by triggering a series of events in HeLa cells including Bax translocation, cytochrome c release, caspase‐3 activation, chromosome fragmentation followed by caspase‐8 activation, Bid cleavage and eventually cell death. Addition of a pan‐caspase inhibitor or overexpression of an anti‐apoptotic protein, Bcl‐xL, prevented OC‐induced cell death. Moreover, OC exhibited a wide anticancer spectrum in multiple cancer cell lines with comparable IC50 values, regardless of the expression levels of HER2 and P‐glycoprotein. In contrast, the IC50 values of three clinical anticancer drugs, etoposide, paclitaxel and vinblastine were significantly elevated in HER2 and/or P‐glycoprotein overexpressing cells. Furthermore, OC showed a similar antitumor effect but lower general toxicity than etoposide against xenografted human tumors in nude mice model. All these data suggested that OC is a promising apoptosis inducer with the potential to be developed into a clinical anticancer drug.

Toxoplasma gondii infection confers resistance against BimS-induced apoptosis by preventing the activation and mitochondrial targeting of pro-apoptotic Bax.

In order to accomplish their life style, intracellular pathogens, including the apicomplexan Toxoplasma gondii, subvert the innate apoptotic response of infected host cells. However, the precise mechanisms of parasite interference with the mitochondrial apoptotic pathway remain unknown. Here, we used the conditional expression of the BH3-only protein BimS to pinpoint the interaction of T. gondii with the intrinsic pathway of apoptosis. Infection of epithelial cells with T. gondii dose-dependently abrogated BimS-triggered release of cytochrome c from host-cell mitochondria into the cytosol, induction of activity of caspases 3, 7 and 9, and chromatin condensation. Furthermore, inhibition of apoptosis in parasite-infected lymphocytes counteracted death of Toxoplasma-infected host cells. Although total cellular levels and mitochondrial targeting of BimS was not altered by the infection, the activation of pro-apoptotic effector proteins Bax and Bak was strongly impaired. Inhibition of Bax and Bak activation by T. gondii was seen with regard to their conformational changes, the cytosol-to-mitochondria targeting and the oligomerization of Bax but not their cellular protein levels. Blockade of Bax and Bak activation was not mediated by the upregulation of anti-apoptotic Bcl-2-like proteins following infection. Further, the BH3-mimetic ABT-737 failed to overcome the Toxoplasma-imposed inhibition of BimS-triggered apoptosis. These results indicate that T. gondii targets activation of pro-apoptotic Bax and Bak to inhibit the apoptogenic function of mitochondria and to increase host-cell viability.

Alpha 5/6 helix domains together with N-terminus determine the apoptotic potency of the Bcl-2 family proteins

A critical process in apoptosis is the permeabilization of the mitochondrial outer membrane (MOM). This process is known to be regulated by the multi-domain Bcl-2 family proteins. For example, the pro-apoptotic proteins Bax and Bak are responsible for forming pores at MOM. The anti-apoptotic proteins (including Bcl-2, Mcl-1 and Bcl-xL), on the other hand, can inhibit this pore-forming process. Interestingly, although these two subgroups of proteins perform opposite apoptotic functions, their structures are very similar. This raises two highly interesting questions: (1) Why do these structurally similar proteins play opposite roles in apoptosis? (2) What are the roles of different functional domains of a Bcl-2 family protein in determining its apoptotic property? In this study, we generated a series of deletion mutants and substitution chimera, and used a combination of molecular biology, bio-informatics and living cell imaging techniques to answer these questions. Our major findings are: (1) All of the Bcl-2 family proteins appear to possess an intrinsic pro-apoptotic property. (2) The N-termini of these proteins play an active role in suppressing their pro-apoptotic function. (3) The apoptotic potency is positively correlated with membrane affinity of the alpha 5/6 helix domains. (4) Charge distribution flanking the alpha 5/6 helices is also important for the apoptotic potency. These findings explain why different members of Bcl-2 family proteins with similar domain composition can function oppositely in the apoptotic process.