| Hepatocellular carcinoma(HCC) is one of the most common malignant tumorsin clinic. Both the morbidity and mortality of HCC have increased in recent years.However, there has been a lack of effective treatment and drugs for HCC. Therefore,searching for new anti-cancer drugs can greatly improve the chance of survival andthe quality of life with liver cancer, which is the key direction of new drug researchand development for major diseases in National five-year plan in recent years.Ling Zhi-8(LZ-8) is a fungal immunomodulatory protein that is isolated frommycelia of Ganoderma lucidium. In anti-tumor research, we found that rLZ-8(recombinant Ganoderma lucidum immunomodulatory protein) could enter and killtumor cells in great quantities without killing for normal tissues.The research grouphad a large number of research which had shown that rLZ-8into the cancer cells is aprerequisite for implementation of the antitumor effects. Therefore, a betterunderstanding of rLZ-8mediated endocytosis mechanism will play a decisive role indeveloping rLZ-8to antitumor medicine.In this study LZ-8coding sequence was optimized according to the Pichiapastoris preference and transferred into the expression vector, pGAPZα A, andelectricity transformed it into the constitutive expression vector X-33strain. Thepositive strains were screened using Zeocin resistant medium, PCR and WesternBlotting. The expression product was separated and purified to obtain high purity ofrLZ-8, It proved to be consistent with The N terminal sequencing of nature LZ-8.In order to research the endocytosis mechanism of rLZ-8in HepG2cells, in thefollowing study, HepG2Cells, treated with Alexa Fluor568(rLZ-8is linked with Alexa Fluor568), were observed to detect the cellular localization of rLZ-8. Weused the pharmacological inhibitors towards the different endocytic pathways. Then,siRNA was used to target the key elements selected in the first round to get furthervalidation.Results:1. Clathrin is not required for rLZ-8entry into HepG2cells.It was found that chlorpromazine, a specific inhibitor of clathrin，did not affectthe efficient rLZ-8internalization, suggesting rLZ-8enters HepG2cells viaclathrin-independent endocytosis.2. rLZ-8enters HepG2cells does not via a caveolae/raft-mediatedendocytosis pathway.It was observed that disruption of membrane rafts by nystatin orcholesterol-sequester progesterone did not inhibit rLZ-8entry into HepG2cells,suggesting rLZ-8enters HepG2cells do not via cholesterol-enriched membranemicrodomains.3. rLZ-8enters HepG2cells via a Src kinase-mediated endocytosispathway.rLZ-8internalization was inhibited by genistein, a selective Src kinase inhibitor,supporting the notion that rLZ-8cell entry is sensitive to Src kinase. In order tofurther confirm, we used siRNA silencing of c-Src, rLZ-8internalization wasinhibited in HepG2cells transfected with siRNA against c-Src. All of thesedemonstrate that rLZ-8entry occurs through a Src kinase-dependent endocytosis.4. rLZ-8enters HepG2cells via the Src-PI3K transduction pathway.It was observed that rLZ-8internalization was partly inhibited by wortmannin,a PI3K inhibitor. C-Src is the upstream molecular of PI3K. All of these suggest thatrLZ-8entry may occur through the Src-PI3K signaling pathway.In conclusion, we successfully expressed and purified the recombinant fungalimmunomodulatory protein rLZ-8, and conducted a study to identify theinternalization mechanism of rLZ-8in HepG2cells. The evidence presented here indicates that rLZ-8entry into HepG2cells occurs by a clathrin-independent,caveolae/raft-independent, Src kinase-dependent, Src-PI3K signaling pathway-dependent endocytosis.Understanding the cellular components involved in rLZ-8entry into the HepG2cells, and a comprehension of the mechanisms that govern this process should openthe possibility of developing anticancer properties of rLZ-8, and the use of LZ-8may be a powerful new strategy for anti-neoplastic therapy. |
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