[Progress of studies on anti-breast tumor mechanism of alkaloids].

Breast tumor has become one of the malignant tumors with the highest incidence, and is a serious threat to human health, especially to women. Chemotherapy is an important anti-breast tumor therapy, which can be used in almost every stage of breast tumor therapy alone or in the combination with surgery and radiation therapy. Alkaloids are a kind of ubiquitous natural products, and important active components of various medicinal plants. A large number of studies have shown that alkaloids could exert an anti-breast tumor effect by inhibiting proliferation, metastasis and angiogenesis, resisting mitosis, promoting apoptosis and autophagy, and triggering cell cycle arrest. The extensive anti-breast tumor effect makes alkaloids an important candidate drug source. This paper reviews the anti-breast tumor mechanism of natural products of alkaloids.

[The role of human lysozyme-like protein 4 in fertilization and its enzymatic properties].

OBJECTIVE: To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties. METHODS: The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods. RESULTS: Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity. CONCLUSIONS: LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.

Efficient production of human goose-type lysozyme 2 in the methylotrophic yeast Pichia pastoris.

Infectious diseases caused by antibiotic multidrug-resistant microorganisms are major causes of morbidity and mortality in humans. Hence, there is an urgent need to search for new antimicrobial agents. Initially known as a defensive effector in the innate immunity of certain organs of the human body, human goose-type lysozyme 2 (hLysG2) has been shown to possess therapeutically useful potential against multidrug-resistant microorganisms. Developing a novel strategy for large-scale production that provides high yields of this protein with high purity, quality, and potency is critical for pharmaceutical applications. To overcome the issues related to prokaryotic expression, here we report the production of recombinant hLysG2 (rhLysG2) using the methylotrophic yeast Pichia pastoris as expression host. The strong inducible alcoholoxidase 1 (AOX1) promoter was used to drive expression of the optimized hLysG2 gene. Under the optimal expression conditions, the lytic activity of rhLysG2 reached 113 U/mL of culture supernatant in shake flask cultivation and this was increased to 2084 U/mL in fed-batch fermentation. Using chitin affinity chromatography and size-exclusion chromatography, rhLysG2 was produced with a yield of 137 mg/L, purity of > 99%, molecular weight of 21,504.6 Da, and specific activity of 13,500 U/mg. In vitro assays indicated that rhLysG2 possessed muramidase activity, isopeptidase activity, and free radical scavenging activity. This report describes an efficient strategy for the production of biologically active rhLysG2 in P. pastoris on a large scale with a high yield, which provides a solid foundation for possible future pharmaceutical applications.

13-Methyl-palmatrubine induces apoptosis and cell cycle arrest in A549 cells in vitro and in vivo.

Corydalis yanhusuo, a well-known herbaceous plant, is commonly used in the treatment of inflammation, injury and pain. One natural agent isolated from Corydalis yanhusuo, 13-methyl-palmatrubine, was found to have a cytotoxic effect on cancer cells as reported in published studies. In the present study, we synthesized a potential anti-lung tumor agent, 13-methyl-palmatrubine and analyzed its activity. 13-Methyl-palmatrubine exhibited a cytotoxic effect on a panel of cancer cell lines in a time- and concentration-dependent manner. Among all the tested cancer cell lines, lung cancer A549 cells were most sensitive to 13-methyl-palmatrubine treatment. Meanwhile 13-methyl-palmatrubine showed less cytotoxicity in human normal cells. Our investigation revealed that 13-methyl­palmatrubine induced apoptosis and cell cycle arrest in A549 cells in a dose-dependent manner. Furthermore, 13-methyl-palmatrubine treatment caused activation of P38 and JNK pathways and blocked the EGFR pathway. In conclusion, our findings demonstrated that 13-methyl-palmatrubine inhibited the growth of A549 cells mediated by blocking of the EGFR signaling pathway and activation of the MAPK signaling pathway and provides a better understanding of the molecular mechanisms of 13-methyl-palmatrubine.

Lobetyol activate MAPK pathways associated with G1/S cell cycle arrest and apoptosis in MKN45 cells in vitro and in vivo.

AIM: The agent lobetyol, which is isolated from Lobelia chinensis, was previously shown to be cytotoxicity againts several cancer cell lines in published report. Today, we perform a study in vitro and in vivo to analyze its anti-carcinoma effect in MKN45 cells and to explore the molecular mechanism. MAIN METHODS: The growth inhibition of lobetyol on MKN45 cells was analyzed with MTT and flow cytometry. Hoechst 33342 staining and TUNEL cover glass staining were used to provide the visual evidence of apoptosis. Western blotting assay was performed to study the activation or blocking of related signaling pathways. KEY FINDINGS: Lobetyol induce apoptosis and cell cycle arrest in a time- and dose-dependent manner in MKN45 cells in our study. This process is mediated by the MAPK signaling pathways. This study confirmed the cytotoxicity of lobetyol in MKN45 cells and provided an insight into the molecular mechanism, which demonstrates the potential of lobetyol as an anti-tumor agent.