Autophagy inhibitors for cancer therapy: Small molecules and nanomedicines.

Autophagy is a conserved process in which the cytosolic materials are degraded and eventually recycled for cellular metabolism to maintain homeostasis. The dichotomous role of autophagy in pathogenesis is complicated. Accumulating reports have suggested that cytoprotective autophagy is responsible for tumor growth and progression. Autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), are promising for treating malignancies or overcoming drug resistance in chemotherapy. With the rapid development of nanotechnology, nanomaterials also show autophagy-inhibitory effects or are reported as the carriers delivering autophagy inhibitors. In this review, we summarize the small-molecule compounds and nanomaterials inhibiting autophagic flux as well as the mechanisms involved. The nanocarrier-based drug delivery systems for autophagy inhibitors and their distinct advantages are also described. The progress of autophagy inhibitors for clinical applications is finally introduced, and their future perspectives are discussed.

Chemo-Phototherapy with Carfilzomib-Encapsulated TiN Nanoshells Suppressing Tumor Growth and Lymphatic Metastasis.

The design of nanomedicine for cancer therapy, especially the treatment of tumor metastasis has received great attention. Proteasome inhibition is accepted as a new strategy for cancer therapy. Despite being a big breakthrough in multiple myeloma therapy, carfilzomib (CFZ), a second-in-class proteasome inhibitor is still unsatisfactory for solid tumor and metastasis therapy. In this study, hollow titanium nitride (TiN) nanoshells are synthesized as a drug carrier of CFZ. The TiN nanoshells have a high loading capacity of CFZ, and their intrinsic inhibitory effect on autophagy synergistically enhances the activity of CFZ. Due to an excellent photothermal conversion efficiency in the second near-infrared (NIR-II) region, TiN nanoshell-based photothermal therapy further induces a synergistic anticancer effect. In vivo study demonstrates that TiN nanoshells readily drain into the lymph nodes, which are responsible for tumor lymphatic metastasis. The CFZ-loaded TiN nanoshell-based chemo-photothermal therapy combined with surgery offers a remarkable therapeutic outcome in greatly inhibiting further metastatic spread of cancer cells. These findings suggest that TiN nanoshells act as an efficient carrier of CFZ for realizing enhanced outcomes for proteasome inhibitor-based cancer therapy, and this work also presents a "combined chemo-phototherapy assisted surgery" strategy, promising for future cancer treatment.

The biomarkers related to immune related adverse events caused by immune checkpoint inhibitors.

The enthusiasm for immune checkpoint inhibitors (ICIs), an efficient tumor treatment model different from traditional treatment, is based on their unprecedented antitumor effect, but the occurrence of immune-related adverse events (irAEs) is an obstacle to the prospect of ICI treatment. IrAEs are a discrete toxicity caused by the nonspecific activation of the immune system and can affect almost all tissues and organs. Currently, research on biomarkers mainly focuses on the gastrointestinal tract, endocrine system, skin and lung. Several potential hypotheses concentrate on the overactivation of the immune system, excessive release of inflammatory cytokines, elevated levels of pre-existing autoantibodies, and presence of common antigens between tumors and normal tissues. This review lists the current biomarkers that might predict irAEs and their possible mechanisms for both nonspecific and organ-specific biomarkers. However, the prediction of irAEs remains a major clinical challenge to screen and identify patients who are susceptible to irAEs and likely to benefit from ICIs.

Efficacy and safety of neoadjuvant immunotherapy in resectable nonsmall cell lung cancer: A meta-analysis.

OBJECTIVE: Progress in neoadjuvant therapy for resectable nonsmall cell lung cancer(NSCLC1)has been stagnant. There have been great achievements in immunotherapy for advanced NSCLC, but the efficacy and safety of neoadjuvant immunotherapy for resectable NSCLC has not been clearly demonstrated. METHODS: Original articles describing the safety and efficacy of neoadjuvant immunotherapy in resectable NSCLC published before January 2020 were retrieved from PubMed, Embase and Cochrane Library. The odds ratio (OR2) and 95 % confidence interval (CI3) were calculated. Heterogeneity and subgroup analysis were performed. RESULTS: A total of 252 patients from seven studies were included. Major pathological response (MPR4) and pathological complete response (pCR5) were used to evaluate the efficacy of neoadjuvant immunotherapy. Compared with neoadjuvant chemotherapy, which exhibited less than 25 % MPR and approximately 2 %-15 % pCR, the values in neoadjuvant immunotherapy were significantly higher (MPR: OR = 0.59; 95 % CI, 0.36-0.98; pCR: OR = 0.16; 95 % CI, 0.09-0.27). Safety was evaluated by the incidence of treatment-related adverse events (TRAE6), surgical resection rate, incidence of surgical complications and surgical delay rate. The pooled OR values of the incidence of TRAE, incidence of surgical complications and surgical delay rate were 0.19, 0.41 and 0.03, respectively, which were significantly better than those for neoadjuvant chemotherapy (95 % CI, 0.04-0.90; 0.22-0.75; 0.01-0.10, respectively). The mean surgical resection rate was 88.70 %, which was similar to the 75 %-90 % rate reported for neoadjuvant chemotherapy (OR = 7.61; 95 % CI, 4.90-11.81). CONCLUSION: Neoadjuvant immunotherapy is effective and safe for resectable NSCLC.

Two New Abietane Diterpenoids from Selaginella moellendorffii Hieron.

Two new abietane diterpenoids, (3S,5R,10S)-3-hydroxy-12-O-demethyl-11-deoxy-19(4→3)-abeo-cryptojaponol, 12,19-dihydroxyabieta-8,11,13-trien-7-one, were isolated from Selaginella moellendorffii Hieron., together with one known abietane diterpenoid and four known tetracyclic triterpenoids. Their structures were characterized by their 1D- and 2D-NMR, ECD and mass spectral studies. All compounds were tested for their inhibitory effects on proliferation of three human cancer cells (human non-small-cell lung carcinoma cell lines A549 and human breast adenocarcinoma cell lines MDA-MB-231 and MCF-7) in vitro. Among them, three compounds displayed modest cytotoxic activities against the above three human cancer cell lines with IC50 values ranging from 16.28 to 40.67 µM.

Design, synthesis, and biological evaluation of ligustrazine - betulin amino-acid/dipeptide derivatives as anti-tumor agents.

The ligustrazine - betulin derivative (TB), TB amino acids derivatives (TB-01 - TB-09) and TB dipeptide derivatives (TB-10 - TB-18) were designed and synthesized. And their in vitro cytotoxic activities were evaluated against four cancer cell lines (Hela, HepG2, BGC-823 and HT-29) and normal cells MDCK by standard methylthiazol tetrazolium (MTT) assay. Most of them demonstrated better antitumor activity than the relevant material betulin. Among them, compound TB-01 showed the best anti-tumor effect on the cancer cells and the lowest toxicity on the normal cells. For example, the cytotoxicity of TB-01 against the cancer cells (mean IC50 = 4.86 ±â€¯1.16 µM) was 3-fold higher than that against the normal cells MDCK (IC50 = 16.11 ±â€¯2.29 µM). Moreover, TB-01 showed better cytotoxic than positive drug cisplatin (DDP) on tumor cells. Besides, the Zebrafish toxicity evaluation test showed that TB-01 demonstrated high biosafety. Subsequently, fluorescent staining, apoptosis detection and cell cycle analysis indicated that TB-01 induced early apoptosis in HepG2 cells and blocked the cell cycle in the G1 phase. In addition, the structure-activity relationships of these derivatives were briefly discussed.

Design, synthesis and biological evaluation of cinnamic acid derivatives with synergetic neuroprotection and angiogenesis effect.

As for complex brain diseases involved with multiple pathogenic factors, it is extremely difficult to achieve curative effect by acting on a single target. Multi-approach drugs provide a promising prospect in the treatment of complex brain diseases and have been attracting more and more interest. Enlightened by synergetic effect of combination in traditional herb medicines, forty-two novel cinnamic acid derivatives were designed and synthesized by introducing capsaicin and/or ligustrazine moieties to enhance biological activities in both neurological function and neurovascular protection. Elevated levels of cell viability on human brain microvascular endothelium cell line (HBMEC-2) and human neuroblastoma cell line (SH-SY5Y) against free radical injury were observed in most of compounds. Among them, compound 14a exhibited the most potent activities with a significant EC50 value of 3.26 ±â€¯0.16 µM (HBMEC-2) and 2.41 ±â€¯0.10 µM (SH-SY5Y). Subsequently, the results of morphological staining and flow cytometry analysis experiments on both cell lines showed that 14a had the potential to block apoptosis, maintain cell morphological integrity and protect physiological function of mitochondria. Moreover, 14a displayed specific angiogenesis effect in the chick chorioallantoic membrane (CAM) assay; and the results of RT-PCR suggested that the mechanism for angiogenesis effect was associated with the enhancement of the expressions of VEGFR2 mRNA in chick embryo. Preliminary structure-activity relationship was analyzed. The above evidences suggested that conjunctures gained by combining active ingredients in traditional herb medicines deserved further study and might provide references in discovering dual-effective lead compounds for brain diseases.

Synthesis and biological activity of glycyrrhetinic acid derivatives as antitumor agents.

Glycyrrhetinic acid (GA) had been the star anticancer lead compound and appealed to many scientists all over the world; however, its antitumor activity was not potent enough. To improve GA's cytoxicity and explore the effect of bonding mode on antitumor activity, 32 compounds including GA-OH series (GO, esters in C-3 position) and GA-NH2 series (GN, with amide linkages in C-3 position) had been designed and synthesized. All the compounds were screened for in vitro cytotoxicity against A549, HepG2, MCF-7, Hela and MDCK cell lines. As a result, all the de-protected (without Boc group) derivatives showed much stronger cytotoxic activity than GA, and surprisingly enough, all the GN series of the compounds were more potent than GO series against various tumor cells. Among them, the compound 26 (amide linkages in C-3 position) exhibited stronger antitumor activity against A549 cell line (IC50 = 2.109 ±â€¯0.11 µM) than the positive drug cisplatin (IC50 = 9.001 ±â€¯0.37 µM). Further studies indicated that compound 26 could induce A549 apoptosis via nuclei fragmentation. The detection of apoptosis and cell cycle analysis indicated that compound 26 could induce the early apoptosis and prevent A549 cells transition from S to G2 phase. Furthermore, the structure-activity relationships were briefly discussed. Among which, current study displayed amide linkages in C-3 position could effectively enhance GA cytotoxicity, providing a new modification strategy for further study.

PSMA-Oriented Target Delivery of Novel Anticancer Prodrugs: Design, Synthesis, and Biological Evaluations of Oligopeptide-Camptothecin Conjugates.

Clinical applications of camptothecin (CPT) have been heavily hindered due to its non-targeted toxicity, active lactone ring instability, and poor water solubility. Targeted drug delivery systems may offer the possibility to overcome the above issues as reported. In this research, a series of prostate-specific membrane antigen (PSMA)-activated CPT prodrugs were designed and synthesized by coupling water-soluble pentapeptide, a PSMA hydrolyzing substrate, to CPT through an appropriate linker. The cytotoxicity of CPT prodrugs was masked temporarily until they were hydrolyzed by the PSMA present within the tumor sites, which restored cytotoxicity. The in vitro selective cytotoxic activities of the prodrugs were evaluated against PSMA-expressing human prostate cancer cells LNCaP-FGC and non-PSMA-expressing cancer cells HepG2, Hela, MCF-7, DU145, PC-3 and normal cells MDCK, LO2 by standard methylthiazol tetrazolium (MTT) assay. Most of the newly synthesized CPT prodrugs showed excellent selective toxicity to PSMA-producing prostate cancer cells LNCaP-FGC with improved water solubility. From among the library, CPT-HT-J-ZL12 showed the best cytotoxic selectivity between the PSMA-expressing and the non-PSMA-expressing cancer cells. For example, the cytotoxicity of CPT-HT-J-ZL12 (IC50 = 1.00 ± 0.20 µM) against LNCaP-FGC (PSMA⁺) was 40-fold, 40-fold, 21-fold, 5-fold and 40-fold, respectively, higher than that against the non-PSMA-expressing cells HepG2 (IC50 > 40.00 µM), Hela (IC50 > 40.00 µM), MCF-7 (IC50 = 21.68 ± 4.96 µM), DU145 (IC50 = 5.40 ± 1.22 µM), PC-3 (IC50 = 42.96 ± 3.69 µM) cells. Moreover, CPT-HT-J-ZL12 exhibited low cytotoxicity (IC50 > 40 µM) towards MDCK and LO2 cells. The cellular uptake experiment demonstrated the superior PSMA-targeting ability of the CPT-HT-J-ZL12, which was significantly accumulated in LNCaP-FGC (PSMA⁺), while it was minimized in HepG2 (PSMA-) cells. Further cell apoptosis analyses indicated that it showed a dramatically higher apoptosis-inducing activity in LNCaP-FGC (PSMA⁺) cells than in HepG2 (PSMA-) cells. Cell cycle analysis indicated that CPT-HT-J-ZL12 could induce cell cycle arrest at the S phase.

Synthesis and biological evaluation of podophyllotoxin derivatives as selective antitumor agents.

To improve podophyllotoxin's cytotoxicity and selective effect, twenty-two podophyllotoxin derivatives had been designed and synthesized. The cytotoxicity of these compounds was evaluated on A549, MCF-7, HepG2 and L-02 cell lines. As a result, most of the compounds were more potent than the positive drugs Etoposide (VP-16) and Doxorubicin which were widely used in clinical for antitumor. There were no magnitude differences about these de-protected (without Boc group) podophyllotoxin amino acid derivatives' cytotoxicity between three tumor cell lines and normal hepatic L-02 cells. Interestingly, some protected (with Boc group) amino acid derivatives and some ligustrazine derivatives showed high selectivity, especially the compound 2 (sarcosine derivative with Boc group). It exhibited highly selectivity both on the cancer cells and the normal cells. The IC50 of compound 2 was 9.5 ±â€¯0.03 nM, 132.6 ±â€¯24.1 nM, 96.4 ±â€¯1.3 nM and 160.2 ±â€¯4.7 nM against A549, MCF-7, HepG2 and L-02 cells, respectively. The SI (IC50L-02/IC50A549) value of compound 2, Doxorubicin and Etoposide was 16.9, 0.2 and 0.5, respectively. Meanwhile, SI (IC50MCF-7/IC50A549) value and SI (IC50HepG2/IC50A549) value of compound 2 were 14.0 and 10.1, respectively. In summary, compound 2 showed high selectivity especially on A549 cells. Further research on cell apoptosis indicated that compound 2 could induce apoptosis of A549 cells through nuclei fragmentation and had lower toxicity to normal hepatic L-02 cells. The detection of apoptosis and cell cycle analysis indicated that compound 2 induced A549 cells apoptosis and prevented A549 cells transition from S to G2 phase while there were no obvious changes on L-02 cells. Moreover, the structure-activity relationships of these derivatives were briefly discussed.

[Construction of Eukaryotic Expression Vector Containing ROP18-ROP12 of Toxoplasma gondii RH Strain].

OBJECTIVE: To construct a recombinant eukaryotic expression plasmid containing ROP18-ROP12 (encoding rhoptry protein 18 and 12) complex gene of Toxoplasma gondii, and examine its expression in eukaryotic cells. METHODS: Recombinant plasmids pVAX1-ROP18 and pVAX1-ROP12 were digested by restriction enzymes BamH I and Xba I . ROP12 gene was cloned into pVAX1-ROP18 to construct the eukaryotic expression plasmid pVAX1-ROP18- ROP12. After colony PCR, enzyme digestion and sequencing, the correct recombinant plasmid pVAX1-ROP18-ROP12 was transfected into HeLa cells. Along with it were groups of empty plasmid, pVAX1-ROP18 and pVAX1-ROP12. Total RNA was extracted from HeLa cells and reverse-transcribed to cDNA. RT-PCR was performed to evaluate mRNA expression of the housekeeping gene ß-actin and ROP18-ROP12 complex gene. Immunofluorescence assay and Western blotting were performed to determine the protein levels of ROP18-ROP12 fusion protein. RESULTS: Colony PCR in recombinant plasmid pVAX1-ROP18-ROP12 showed a specific band at about 2 373 bp, consistent with expectation. The extracted recombinant plasmids were confirmed by Hind III, BamH I and Xba I digestion. Sequencing results showed that the sequence of pVAX1-ROP18-ROP12 was 100% identical to that of T. gondii RH strain ROP18 gene (Accession No. AM075204.1) and 99% identical to that of T. gondii RH strain ROP12 gene (Accession No. DQ096559.1). Further, RT-PCR showed amplification products at 613 bp for ß-actin in all the groups, while only the pVAX1-ROP18-ROP12 transfection group showed amplification products for the ROP18-ROP12 complex at 2,373 bp. In addition, the indirect immunofluorescence assay showed yellow-green fluorescence in HeLa cells transfected with pVAX1-ROP18-ROP12, but not in control cells. Western blotting showed that the ROP18-ROP12 fusion protein was expressed in HeLa cells transfected with recombinant plasmid pVAX1-ROP18-ROP12. CONCLUSIONS: The recombinant eukaryotic plasmid pVAX1-ROP18-ROP-2 is constructed and can be expressed in eukaryotic system.

Regulation of pepc gene expression in Anabaena sp. PCC 7120 and its effects on cyclic electron flow around photosystem I and tolerances to environmental stresses.

Since pepc gene encoding phosphoenolpyruvate carboxylase (PEPCase) has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregulated (forward) or downregulated (reverse) pepc gene in Anabaena sp. PCC 7120. Results from real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and enzymatic analysis showed that PEPCase activity was significantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I (PSI) activity was inhibited, as reflected by Y(I), and Y(NA) was elevated, and dark reduction of P700(+) was stimulated, indicating enhanced cyclic electron flow (CEF) around PSI in the reverse mutant. Additionally, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.

Recombinant expression and functional analysis of a Chlamydomonas reinhardtii bacterial-type phosphoenolpyruvate carboxylase gene fragment.

To investigate the function of a bacterial-type phosphoenolpyruvate carboxylase (PEPC2) derived from photosynthetically-grown Chlamydomonas reinhardtii, a fragment of the pepc2 gene was cloned and expressed in Escherichia coli. After optimal induction for 6 h, PEPC activity in the reverse mutant was lower than wild type (0.9 vs. 1.7 U/mg protein), and soluble protein was also lower than wild type (119 vs. 186 mg/g dry wt). In contrast, the total lipid content was increased from 56 (in wild type) to 71 mg/g dry wt, despite the growth rate being slightly diminished. The changes in PEPC activity, soluble protein and total lipid in the forward mutant were the opposite (2.4 U/mg, 230 mg/g, and 44 mg/g dry wt, respectively). Together, these data indicate that PEPC may function as a metabolic pivot in the regulation of protein and lipid accumulation in this alga.

The validation and application of the Chinese version of perceived nursing work environment scale.

AIMS AND OBJECTIVES: To improve the development of the Chinese version of Perceived Nursing Work Environment (C-PNWE) scale by examination and application and to explore the nurses' perception of their working environment in a hospital. BACKGROUND: The C-PNWE scale was translated and revised from the PNWE scale. The least of perfection is that the development of C-PNWE ignored that the psychometric properties of the PNWE instrument were established of critical care nurses and further application and testing of the PNWE in various patient care settings were recommended. DESIGN: This is a cross-sectional design. Nurses from different departments of a hospital were sampled by convenience sampling and investigated by self-administrated questionnaire. METHODS: Data obtained through questionnaires were analysed by descriptive statistical analyses and profile analyses using the Statistical Package for the Social Sciences (SPSS) Chinese version 17.0 software. RESULTS: The coincident and level profile analyses indicated that these groups can merge into one group, and the profile of measurement result of this merged group would not exhibit flatness. Among six dimensions of C-PNWE scale, the Staffing and Resource Adequacy got the lowest average score. Among 41 items, 'Opportunity for staff nurse to participate in policy decisions' got the lowest mean. CONCLUSIONS: The C-PNWE scale shows good psychometric properties and can be used to explore nurses' perspectives of the nursing practice environment in China. And the situation of nurses' perceived working environment in China needs further study. RELEVANCE TO CLINICAL PRACTICE: Shaping nursing practice environments to promote desired outcomes requires valid and reliable measures to assess practice environments prior to, during and following efforts to implement change. The C-PNWE scale can be a useful measurement tool for administrators to improve the nursing work environment in China.

[Cloning and bioinformatics analysis of rhoptry protein 11 of Toxoplasma gondii].

Total RNA was extracted from tachyzoites of RH strain of Toxoplasma gondii. The open reading frame of ROP11 gene was amplified by using a pair of specific primers designed according to the coding sequence of ROP11 gene (Accession No. DQ077905). The RT-PCR product was digested by restriction enzyme EcoR I and Not I, and then ligated into a pGEX-6P-2 vector. The recombinant plasmid was transferred into E. coli XL-Blue. The positive clones was selected by colony PCR, and confirmed by the double restriction enzyme digestion and sequencing. The RT-PCR product was 1,548 bp. The recombinant plasmid was confirmed by colony PCR and double restriction enzyme digestion. Sequencing results showed that the obtained ROP11 gene was 1 548 bp (Accession No. KC456639). There was a high sequence consistency (99%) between the obtained ROP11 gene sequence and the Toxoplasma ROP11 gene from GenBank. Bioinformatics analysis showed that the ROP11 protein (Mr 57,020) consisted of the signal peptide (amino acids 1-26), 12 conservative domains, a serine/threonine protein kinase catalytic domain (amino acids 170-511), and two potential N-glycosylation sites.

HPV16 CTL epitope peptide-activated dendritic cell and natural killer co-culture for therapy of cervical cancer in an animal model.

There is increasing evidence that natural killer (NK) cells play an important role in antitumor immunity following dendritic cell (DC) vaccination. Little is known, however, about the optimal stimulation of DCs by epitopes and NK interactions for cytotoxicity in tumors. In this study, DC cells activated by the HPV16E7.49-57 epitope and LPS were co-cultured with NK cells in vitro, and then used ot immunize mice to study CTL activity of TC-1, which constitutively expresses HPV16E6E7, with an LDH release assay. Cytotoxicity in mice immunized with DC loaded with epitope HPVE7.49-57 vaccine co-cultured with NK was enhanced significantly (p<0.01). In conclusion, talk-across between DC and NK cells enhances their functions, also improving cytotoxicity againsttumor cells, suggesting that activated DC-NK by epitopes has potential application for cancer-specific immuno-cellular therapy.

[Expression of EGF and SP domain proteins of MASP-2 and preparation of their polyclonal antibodies].

AIM: To clone and prokaryotically express the EGF and SP domain proteins of human mannan-binding lectin associated serine protease-2 (MASP-2), and prepare their polyclonal antibodies and analyze their immunogenicity. METHODS: The cDNA of human MASP-2 was transcripted reversely from total RNA extracted from human fetal liver tissue, and then EGF and SP domain genes of MASP-2 were amplified from the cDNA by PCR. The EGF and SP fragment domain genes were cloned into pGEX-6P-2 expression vector and induced by IPTG to express GST fusion protein. The BALB/c mice were immunized with the purified fusion proteins to generate polyclonal antibodies. The specificity of the polyclonal antibodies was analysed with Western blotting, and the titer was determined by indirect ELISA. RESULTS: We successfully constructed the recombinant pGEX-6P-2-EGF and pGEX-6P-2-SP and induced the expressions of the GST-EGF and GST-SP fusion proteins. The specificity of the polyclonal antibodies was higher, and had no cross reaction with other proteins. Indirect ELISA showed that the titer of anti-EGF antibody was more than 1:32 000 and anti-SP antibody was more than 1:40 000. CONCLUSION: The polyclonal antibodies of the EGF and SP domain proteins we successfully obtained were of higher specificity and titer, which provides the basis for further research on MASP-2.

[Cloning, expression and immunoreactivity analysis of rhoptry protein 18 (rop18) from Toxoplasma gondii].

OBJECTIVE: To clone and express rhoptry protein 18 (ROP18) gene of Toxoplasma gondii, and analyze its immunoreactivity. METHODS: The genomic DNA was extracted from T. gondii (RH strain) tachyzoites. TgROP18 gene was amplified by PCR, and cloned into pET30a (+) vector. The constructed pET30a (+)-TgROP18 was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. The recombinant protein was analyzed through SDS-PAGE, and identified by Western blotting with mouse anti-T. gondii serum. RESULTS: The TgROP18 gene was about 1 665 bp in length and encoded for a protein of 544 amino acid residues and the former 47 amino acids consisted signal peptide sequences. PCR, enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET30a (+)-TgROP18 was constructed. Bacteria containing recombinant plasmid pET30a (+)-TgROP18 expressed a soluble protein of His-TgROP18 (M, 59 800) after being induced with IPTG. His-TgROP18 reacted positively with mouse anti-T. gondii serum by Western blotting analysis. CONCLUSION: The soluble His-TgROP18 protein shows certain immunoreactivity.

[Effect of rapamycin on proliferation, apoptosis and regulation of chemokine receptor CXCR4 in RPMI8226 cells].

This study was purposed to investigate the effect of rapamycin on proliferation, apoptosis, cell cycle progression and the regulation of chemokine receptor CXCR4 on RPMI8226 cells. Different concentrations of rapamycin were used to treat the multiple myeloma cell line RPMI8226 for different times. The proliferation of the cells was detected by MTT assay; the apoptosis rate and cell cycle were determined by flow cytometry (FCM); apoptosis of cells was observed by inverted microscopy; the cylin D1, CXCR4 and mTOR mRNA expressions were detected by RT-PCR or FQ-PCR after treating RPMI8226 cells with different concentrations of rapamycin. The results indicated that the rapamycin could inhibit the proliferation of RPMI8226 cells and induce their apoptosis. The cell cycle was arrested at the G(0)/G(1) phase. PCR results showed the down-regulation of mTOR, cyclin D1 and mTOR mRNA expressions after treating RPMI8226 cells with different concentrations of rapamycin for 24 hours. It is concluded that the rapamycin significantly inhibits the growth of RPMI8226 cells in a dose-and time-dependent mannes and induce cell apoptosis. Cell cycle arrests at the G(0)/G(1) phase, may be due to the down-regulation of the mTOR and cyclin D1 expressions. In additions, the down-regulation of CXCR4 mRNA expression is correlated with the reduction of adhesion between myeloma cells and stromal cells.