Targeted treatment for biofilm-based infections using PEGylated tobramycin.

Chronic infections often involve biofilm-based bacteria, in which the biofilm results in significant resistance against antimicrobial agents and prevents eradication of the infection. The physicochemical barrier presented by the biofilm matrix is a major impediment to the delivery of many antibiotics. Previously, PEGylation has been shown to improve antibiotic penetration into biofilms in vitro. In these studies, PEGylating tobramycin was investigated both in vitro and in vivo. Two distinct PEGylated tobramycin molecules were synthesized (mPEG-SA-Tob and mPEG-AA-Tob). Then, in a P. aeruginosa biofilm in vitro model, we found that mPEG-SA-Tob can operate as a prodrug and showed 7 times more effectiveness than tobramycin (MIC80: 14 µM vs.100 µM). This improved biofilm eradication is attributable to the fact that mPEG-SA-Tob can aid tobramycin to penetrate through the biofilm and overcome the alginate-mediated antibiotic resistance. Finally, we used an in vivo biofilm-based chronic pulmonary infection rat model to confirm the therapeutic impact of mPEG-SA-Tob on biofilm-based chronic lung infection. mPEG-SA-Tob has a better therapeutic impact than tobramycin in that it cannot only stop P. aeruginosa from multiplying in the lungs but can also reduce inflammation caused by infections and prevent a recurrence infection. Overall, our findings show that PEGylated tobramycin is an effective treatment for biofilm-based chronic lung infections.

Viability of Lactobacillus acidophilus in Thin-Film Freeze-Dried Powders Filled in Delayed-Release Vegetarian Capsules in a Simulated Gastric Fluid.

Previously, we have shown that thin-film freeze-drying can be applied to prepare dry powders of bacteria such as Lactobacillus acidophilus. Herein, we tested the viability of L. acidophilus in thin-film freeze-dried powders (TFF powders) filled in delayed-release vegetarian capsules in a simulated gastric fluid (SGF) consisting of 0.1N hydrochloric acid and sodium chloride. Initially, we determined the water removal rate from frozen thin films on relatively larger scales (i.e., 10-750 g). We then prepared and characterized two TFF powders of L. acidophilus with either sucrose and maltodextrin or sucrose and hydroxypropyl methylcellulose acetate succinate (HPMC-AS), a pH-sensitive polymer, as excipients and evaluated the viability of the bacteria after the TFF powders were filled in delayed-release vegetarian capsules and the capsules were incubated in the SGF for 30 min. On 10-750 g scales and at the settings specified, water removal from frozen thin films was faster than from slow shelf-frozen bulk solids. When the L. acidophilus in sucrose and HPMC-AS TFF powder was filled into a delayed-release capsule that was placed into another delayed-release capsule, the bacterial viability reduction after incubation in the SGF can be minimized to within 1 log in colony forming unit (CFU). However, for the L. acidophilus in sucrose and maltodextrin TFF powder, even in the capsule-in-capsule dosage form, bacterial CFU reduction was > 2 logs. TFF powders of live microorganisms containing an acid-resistant material in capsule-in-capsule delayed-release vegetarian capsules have the potential for oral delivery of those microorganisms.

Targeted treatment of triple-negative-breast cancer through pH-triggered tumour associated macrophages using smart theranostic nanoformulations.

Triple-negative breast cancer (TNBC) represents 15-25 % of the new breast cancer cases diagnosed worldwide every year. TNBC is among the most aggressive and worst prognosis breast cancer, mainly because targeted therapies are not available. Herein, we developed a magnetic theranostic hybrid nanovehicle for targeted treatment of TNBC through pH-triggered tumour associated macrophages (TAMs) targeting. The lipid core of the nanovehicle was composed of a Carnaúba wax matrix that simultaneously incorporated iron oxide nanoparticles and doxorubicin (DOX) - a chemotherapeutic drug. These drug-loaded wax nanovehicles were modified with a combination of two functional and complementary molecules: (i) a mannose ligand (macrophage targeting) and (ii) an acid-sensitive sheddable polyethylene glycol (PEG) moiety (specificity). The TAMs targeting strategy relied on the mannose - mannose receptor recognition exclusively after acid-sensitive "shedding" of the PEG in the relatively low tumour microenvironment pH. The pH-induced targeting capability towards TAMs was confirmed in vitro in a J774A.1 macrophage cell line at different pH (7.4 and 6.5). Biocompatibility and efficacy of the final targeted formulations were demonstrated in vitro in the TNBC MDA-MB-231 cell line and in vivo in an M-Wnt tumour-bearing (TNBC) mouse model. A preferential accumulation of the DOX-loaded lipid nanovehicles in the tumours of M-Wnt-tumour bearing mice was observed, which resulted both on an efficient tumour growth inhibition and a significantly reduced off-target toxicity compared to free DOX. Additionally, the developed magnetic hybrid nanovehicles showed outstanding performances as T2-contrast agents in magnetic resonance imaging (r2 ≈ 400-600 mM-1·s-1) and as heat generating sources in magnetic hyperthermia (specific absorption rate, SAR ≈ 178 W·g-1Fe). These targeted magnetic hybrid nanovehicles emerge as a suitable theranostic option that responds to the urgent demand for more precise and personalized treatments, not only because they are able to offer localized imaging and therapeutic potential, but also because they allow to efficiently control the balance between safety and efficacy.

Aerosolizable Plasmid DNA Dry Powders Engineered by Thin-film Freezing.

PURPOSE: This study was designed to test the feasibility of using thin-film freezing (TFF) to prepare aerosolizable dry powders of plasmid DNA (pDNA) for pulmonary delivery. METHODS: Dry powders of pDNA formulated with mannitol/leucine (70/30, w/w) with various drug loadings, solid contents, and solvents were prepared using TFF, their aerosol properties (i.e., mass median aerodynamic diameter (MMAD) and fine particle fraction (FPF)) were determined, and selected powders were used for further characterization. RESULTS: Of the nine dry powders prepared, their MMAD values were about 1-2 µm, with FPF values (delivered) of 40-80%. The aerosol properties of the powders were inversely correlated with the pDNA loading and the solid content in the pDNA solution before TFF. Powders prepared with Tris-EDTA buffer or cosolvents (i.e., 1,4-dioxane or tert-butanol in water), instead of water, showed slightly reduced aerosol properties. Ultimately, powders prepared with pDNA loading at 5% (w/w), 0.25% of solid content, with or without Tris-EDTA were selected for further characterization due to their overall good aerosol performance. The pDNA powders exhibited a porous matrix structure, with a moisture content of < 2% (w/w). Agarose gel electrophoresis confirmed the chemical integrity of the pDNA after it was subjected to TFF and after the TFF powder was actuated. A cell transfection study confirmed that the activity of the pDNA did not change after it was subjected to TFF. CONCLUSION: It is feasible to use TFF to produce aerosolizable pDNA dry powder for pulmonary delivery, while preserving the integrity and activity of the pDNA.

Accelerated water removal from frozen thin films containing bacteria.

Freeze-drying, or lyophilization, is widely used to produce pharmaceutical solids that contain temperature-sensitive materials. Herein, using Escherichia coli as a model live organism, whose viability in dry powders is highly sensitive to the water content in the powders, we demonstrated that the drying rate from the frozen thin films generated by thin-film freezing (TFF) is significantly faster than from the bulk frozen solids in conventional shelf freeze-drying. This is likely because the loosely stacked frozen thin films provided a larger solid-air interface and the low thickness of the thin films provided a low mass transfer resistance. The highly porous microstructure and high specific surface area of the thin-film freeze-dried powders may also be related to the faster drying observed. Moreover, we demonstrated that TFF can be applied to produce dry powders of E. coli, a Gram-negative bacterium, or Lactobacillus acidophilus, a Gram-positive bacterium, with minimum bacterial viability loss (i.e., within one log reduction). It is concluded that the TFF technology is promising in accelerating water removal from frozen samples.

Apoptotic body-inspired nanoparticles target macrophages at sites of inflammation to support an anti-inflammatory phenotype shift.

Chronic inflammation is a significant pathological process found in a range of disease states. Treatments to reduce inflammation in this family of diseases may improve symptoms and disease progression, but are largely limited by variable response rates, cost, and off-target effects. Macrophages are implicated in many inflammatory diseases for their critical role in the maintenance and resolution of inflammation. Macrophages exhibit significant plasticity to direct the inflammatory response by taking on an array of pro- and anti-inflammatory phenotypes based on extracellular cues. In this work, a nanoparticle has been developed to target sites of inflammation and reduce the inflammatory macrophage phenotype by mimicking the anti-inflammatory effect of apoptotic cell engulfment. The nanoparticle, comprised of a poly(lactide-co-glycolide) core, is coated with phosphatidylserine (PS)-supplemented cell plasma membrane to emulate key characteristics of the apoptotic cell surface. The particle surface is additionally functionalized with an acid-sensitive sheddable polyethylene glycol (PEG) moiety to increase the delivery of the nanoparticles to low pH environments such as those of chronic inflammation. In a mouse model of lipopolysaccharide-induced inflammation, particles were preferentially taken up by macrophages at the site and promoted an anti-inflammatory phenotype shift. This PEGylated membrane coating increased the delivery of nanoparticles to sites of inflammation and may be used as a tool alone or as a delivery scheme for additional cargo to reduce macrophage-associated inflammatory response.

Pulmonary biofilm-based chronic infections and inhaled treatment strategies.

Certain pulmonary diseases, such as cystic fibrosis (CF), non-CF bronchiectasis, chronic obstructive pulmonary disease, and ventilator-associated pneumonia, are usually accompanied by respiratory tract infections due to the physiological alteration of the lung immunological defenses. Recurrent infections may lead to chronic infection through the formation of biofilms. Chronic biofilm-based infections are challenging to treat using antimicrobial agents. Therefore, effective ways to eradicate biofilms and thus relieve respiratory tract infection require the development of efficacious agents for biofilm destruction, the design of delivery carriers with biofilm-targeting and/or penetrating abilities for these agents, and the direct delivery of them into the lung. This review provides an in-depth description of biofilm-based infections caused by pulmonary diseases and focuses on current existing agents that are administered by inhalation into the lung to treat biofilm, which include i) inhalable antimicrobial agents and their combinations, ii) non-antimicrobial adjuvants such as matrix-targeting enzymes, mannitol, glutathione, cyclosporin A, and iii) liposomal formulations of anti-biofilm agents. Finally, novel agents that have shown promise against pulmonary biofilms as well as traditional and new devices for pulmonary delivery of anti-biofilm agents into the lung are also discussed.

Novel formulations and drug delivery systems to administer biological solids.

Recent advances in formulation sciences have expanded the previously limited design space for biological modalities, including peptide, protein, and vaccine products. At the same time, the discovery and application of new modalities, such as cellular therapies and gene therapies, have presented formidable challenges to formulation scientists. We explore these challenges and highlight the opportunities to overcome them through the development of novel formulations and drug delivery systems as biological solids. We review the current progress in both industry and academic laboratories, and we provide expert perspectives in those settings. Formulation scientists have made a tremendous effort to accommodate the needs of these novel delivery routes. These include stability-preserving formulations and dehydration processes as well as dosing regimes and dosage forms that improve patient compliance.

Aerosolizable siRNA-encapsulated solid lipid nanoparticles prepared by thin-film freeze-drying for potential pulmonary delivery.

Lipid nanoparticles are increasingly used for drug and gene delivery, including the delivery of small interfering RNA (siRNA). Pulmonary delivery of drug molecules carried by lipid nanoparticles directly into the lung may improve the treatment of certain lung diseases. The present study was designed to test the feasibility of engineering aerosolizable dry powder of lipid nanoparticles by thin-film freeze-drying (TFFD). Solid lipid nanoparticles (SLNs) comprised of lecithin, cholesterol, and a lipid-polyethylene glycol conjugate were prepared by solvent evaporation. Dry powders of the SLNs were prepared by TFFD, spray drying, or conventional shelf freeze-drying. The physical and aerosol properties of the dry powders as well as the physical properties of the SLNs reconstituted from the dry powders were evaluated. The particle size, polydispersity index, and the zeta potential of the SLNs were preserved after they were subjected to TFFD and reconstitution, but not after they were subjected to conventional shelf freeze-drying and reconstitution, and the dry powder prepared by TFFD showed better aerosol performance properties than that prepared by spray drying. SLNs encapsulated with siRNA can also be successfully transformed into aerosolizable dry powder by TFFD, and subjecting the siRNA-encapsulated SLNs to TFFD did not negatively affect the function of the siRNA. It is concluded that TFFD represents a promising method to prepare aerosolizable dry powder of lipid nanoparticles.

The COVID-19 Vaccine Race: Challenges and Opportunities in Vaccine Formulation.

In the race for a safe and effective vaccine against coronavirus disease (COVID)-19, pharmaceutical formulation science plays a critical role throughout the development, manufacturing, distribution, and vaccination phases. The proper choice of the type of vaccine, carrier or vector, adjuvant, excipients, dosage form, and route of administration can directly impact not only the immune responses induced and the resultant efficacy against COVID-19, but also the logistics of manufacturing, storing and distributing the vaccine, and mass vaccination. In this review, we described the COVID-19 vaccines that are currently tested in clinical trials and provided in-depth insight into the various types of vaccines, their compositions, advantages, and potential limitations. We also addressed how challenges in vaccine distribution and administration may be alleviated by applying vaccine-stabilization strategies and the use of specific mucosal immune response-inducing, non-invasive routes of administration, which must be considered early in the development process.

Studies of Thioamide Effects on Serine Protease Activity Enable Two-Site Stabilization of Cancer Imaging Peptides.

Thioamide substitutions in peptides can be used as fluorescence quenchers in protease sensors and as stabilizing modifications of hormone analogs. To guide these applications in the context of serine proteases, we here examine the cleavage of several model substrates, scanning a thioamide between the P3 and P3' positions, and identify perturbing positions for thioamide substitution. While all serine proteases tested were affected by P1 thioamidation, certain proteases were also significantly affected by other thioamide positions. We demonstrate how these findings can be applied by harnessing the combined P3/P1 effect of a single thioamide on kallikrein proteolysis to protect two key positions in a neuropeptide Y-based imaging probe, increasing its serum half-life to >24 h while maintaining potency for binding to Y1 receptor expressing cells. Such stabilized peptide probes could find application in imaging cell populations in animal models or even in clinical applications such as fluorescence-guided surgery.

Determination of Endocrine Disruption Potential of Bisphenol A Alternatives in Food Contact Materials Using In Vitro Assays: State of the Art and Future Challenges.

Alternatives to bisphenol A (BPA) are developed for food contact materials as a result of increasing evidence of exposure-correlated harmful effects of BPA. In vitro assays provide the fast, affordable, and mechanism insightful ways to screen endocrine disruption (ED), which is a major concern of new BPA alternatives. In this review, we summarize the safety and regulation information on the alternatives to BPA, review the state of the art of in vitro assays for ED evaluation, highlight their advantages and limitations, and discuss the challenges and future research needs. Our review shows that ligand binding, reporter gene, cell proliferation, and steroidogenesis are four commonly used in vitro assays to determine the ED at the response of receptor, gene transcription, and whole cell level. Major challenges are found from in vitro-in vivo translation and identification of ED chemicals in polymers. More studies on these areas are needed in the future.

[The Concentration Effect of Near-Infrared Quantum Cutting Luminescence of Tm(3+) Ion Sensitized with Bi(3+) Ion in YNbO(4) Phosphor].

Searching for new energy source is one of the most important projects faced by the global, while the most ideal new energy source is solar cell. Near infrared quantum cutting luminescence method can doubly transfer large energy photon which is not sensitive to Si or Ge solar cell to small energy photon which is sensitive to Si or Ge solar cell. It can resolve the spectral mismatch problem and largely enhance solar cell efficiency. Therefore, it is significant. The concentration effect of near-infrared quantum cutting luminescence of Tm3+Bi3+∶YNbO4 phosphor is reported in present manuscript. Through the measurement of excitation and emission spectra, it is found that the Tm0.058Bi0.010Y0.932NbO4 powder phosphor has intense 1 820.0 nm near-infrared quantum cutting luminescence. Further analysis finds they are multi-photon quantum cutting luminescence induced by the cross-energy transfer process. The population of 1G4 energy level may be directly transferred to lower energy level mainly through {1G4­3H4, 3H6­3H5} and {1G4­3H5, 3H6­3H4} cross-energy transfer processes, i. e. one population of the 1G4 energy level may effectively lead to two populations, which are positioned at the 3H4 and 3H5 energy levels, respectively, mainly through {1G4­3H4, 3H6­3H5} and {1G4­3H5, 3H6­3H4} cross-energy transfer processes. This may also effectively lead to three populations of the 3F4 energy level through {3H4­3F4, 3H6­3F4} cross-energy transfer process from the 3H4 level and multi-phonon non-radiative relaxation from the 3H5 level, respectively. This results in the effective three-photon near-infrared quantum cutting of the 3F4­3H6 fluorescence of Tm3+ ion. It's also found that the sensitization action of Bi3+ ion to Tm3+ ion is very strong. The enhancement of the 1 820.0 nm near-infrared quantum cutting luminescence, of Tm0.058Bi0.010Y0.932NbO4 relative to Tm0.005Y0.995NbO4, is about 175.5 times, when excited by the 302.0 nm light. The present results are significant for the exploration of the next-generation multi-photon near-infrared quantum cutting germanium solar cell.

Characterization of the 5' flanking region of lipase gene from Penicillium expansum and its application in molecular breeding.

A major challenge for further promotion of lipase productivity in Penicillium expansum PE-12 is to find a suitable promoter that can function efficiently in this industrial strain. In this study, the 5' flanking region of P. expansum lipase (Ppel) containing a putative novel promoter sequence was characterized by fusing to ß-glucuronidase (GUS) and subsequently introducing into P. expansum. As a result, all the transformants showed blue color quickly after incubation in GUS detection buffer, suggesting a strong promoter activity of this fragment. Glucose repression was identified for the promoter, whereas olive oil acted as a positive regulator. Facilitated by this novel promoter, P. expansum PE-12 was genetically modified, with an improved lipase yield, via a recombinant plasmid with P. expansum lipase gene (PEL) under the control of Ppel promoter and TtrpC terminator. The highest lipase yield among the modified strains could attain 2,100 U/mL, which is more than twofold of the previous industrial strain (900 U/mL). The engineered strain through molecular breeding method as well as this new promoter has great value in lipase industry.

[Influence of phosphoprotein associated with glycosphingolipid microdomains 1 on biologic behavior of human prostatic cancer cell line in-vitro].

OBJECTIVE: To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8. METHODS: The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected, respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC) was used for the staining of intracellular F-actin, Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin. RESULTS: After IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAG1-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21(WAF1/CIP1) and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however, the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma, correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma. CONCLUSIONS: An over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.

[Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro].

OBJECTIVE: To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro. METHODS: Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells. RESULTS: A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1E8. Western blot revealed that PAG1 protein was downregulated in PC-3M-1E8 cell line which was in agreement with the gene expression at mRNA level. The proliferation ability and clonogenicity of PAG1 overexpression cells by stable transfection were markedly decreased in comparing with that of the control cells (P < 0.05). Colonies formed in stably PAG1-transfected cells, the vector-transfected clones and parental cells were 26.7 ± 5.2, 47.2 ± 3.2 and 52.3 ± 3.4 respectively (P < 0.05). Flow cytometry analysis showed that the stable PAG1-transfected cells at G0-G1 phase were significantly more than that of the control cells (P < 0.05). However, no difference of the apoptosis rate was found between PAG1-transfected cells and control cells (P > 0.05). The number of cells passing through the matrigel and multipore membrane was also decreased in the stable PAG1-transfected cells (35.1 ± 4.9) compared with those of the vector-transfected clones (127.6 ± 6.6) and parental cells (135.0 ± 5.0, P < 0.05). CONCLUSIONS: Using cDNA microarray technique and differential gene expression analysis of sublines of the parental cancer cell lines enable of revealing the metastasis-related genes, among which PAG1 represents one of those under-expressed genes in the high metastatic subline PC-3M-1E8. Transfection expression of PAG1 suppresses cell proliferation, anchorage-independent growth and invasive ability of PC-3M-1E8 cells in vitro. Conclusively, PAG1 may play an important role in inhibiting the proliferation, invasion and metastasis of the cancer cells.

[Overexpression of human tumor metastasis-related gene TMSG-1 suppresses cell proliferation and invasion of a highly metastatic prostate cancer cell line PC-3M-1E8 in vitro].

OBJECTIVE: To investigate the effects of tumor metastasis-related gene TMSG-1 overexpression on the proliferation and invasion of a highly metastatic prostate cancer cell line in vitro. METHODS: The eukaryotic expression plasmids containing full-length TMSG-1 cDNAs were stably transfected into the highly metastatic prostate cancer cell line PC-3M-1E8. Clones highly expressing TMSG-1 were identified by RT-PCR and Western Blot analysis after G418 screening. The cell proliferation was detected by cell growth curve, MTT assay and soft agar colony formation assay. The invasive potential of tumor cells in vitro was tested by Matrigel invasion assay. RESULTS: Three TMSG-1 overexpression clones were selected. Cell growth curve and MTT assay showed that TMSG-1 overexpression clones exhibited a strong inhibition of proliferation compared with that of the parental cells or those transfected with vector alone from the third day of culture (P <0.05). In vitro analysis also showed that the TMSG-1 transfected clones exhibited a decreased clonogenicity in soft agar compared with that of the parental cells or those transfected with vector only (P < 0.05). TMSG-1 expression significantly suppressed cell invasion in vitro of TMSG-1-transfected PC-3M-IE8 cells (P < 0.05). CONCLUSION: The TMSG-1 protein may serve as a tumor metastasis suppressor due to inhibiting cell proliferation and invasion of the highly metastatic prostate cancer cell line PC-3M-1E8.

[Correlation of expression of RhoC with invasiveness of prostate cancer cell line PC-3M in vitro].

OBJECTIVE: To investigate the expression of RhoC in prostate cancer cells with different metastatic potential and the correlation with invasiveness. METHODS: Expression of RhoC mRNA and protein in human prostate cancer cell subclones PC-3M-2B4 with non-metastatic potential and PC-3M-1E8 with highly metastatic potential was detected by RT-PCR and Western blot. Eukaryotic expression plasmids of RhoC were constructed and transfected into PC-3M-2B4. The biological effects were observed, including in vitro invasion by Boyden chamber assay, motility by would healing assay, alteration of microfilament network by the staining of TRTIC-phalloidin, activities of matrix metalloproteinases (MMP) by gelatin zymography analysis and expression of p-Akt by Western blot assay. RESULTS: The expression levels of RhoC mRNA and protein varied in the two different metastatic subclones of human prostate cancer cell. RhoC was significantly upregulated in the highly metastatic subclones in comparison to the nonmetastatic counterpart (P < 0.01). As shown in Boyden chamber assay, the invasive capacity of transfected cells overexpressing RhoC was significantly promoted as reflected by more penetrating cells (125.21 +/- 10.43) than the antisense transcripts (46.22 +/- 8.12), the negative (53.77 +/- 8.56) and blank controls (57.68 +/- 7.25). Further study by would healing assay indicated that cells overexpressing RhoC were more motile in actin-based active movement (would healing ratio after 16 h of the sense transcripts, antisense transcripts, negative controls and blank controls was 62.38 +/- 2.36, 29.47 +/- 1.86, 32.23 +/- 2.43, 31.88 +/- 2.67 respectively). The TRITC-phalloidin staining revealed less actin filament bundles and a fuzzy network of shorter filaments in the sense transcripts. In addition, MMP-2 activity and p-Akt expression level were upregulated in the sense transcripts. CONCLUSION: RhoC overexpression could promote the invasive capacity of human prostate cancer cell in vitro and it's expression level correlated positively with the metastatic capacity of human prostate cancer cell, so RhoC may be a potential target in the development of a novel strategy for treating metastasis of prostate cancer.

[Overexpression of tumor metastasis suppressor gene 1 suppresses proliferation and invasion, but enhances apoptosis of human breast cancer cells MDA-MB-231 cells].

OBJECTIVE: To investigate the effects of tumor metastasis suppressor gene 1 (TMSG-1) overexpression on the proliferation, invasion and apoptosis of breast cancer cells and to determine possible correlations of TMSG-1 and metastasis of breast cancer. METHODS: Full-length human TMSG-1 coding sequences were cloned into plasmid pcDNA3.0-FLAG. The recombinant plasmids constructs were transfeced into MDA-MB-231, a highly malignant breast cancer cell line. Parental, vector-only stable transfectant and TMSG-1 stable transfectant clones were tested by MTT, soft agar colony formation and Boyden chamber assays. At twenty-four hours and forty-eight hours post transient transfection, double staining with Annexin-V-FITC and PI were employed to distinguish apoptotic cells from living cells by flow cytometry analysis. RESULTS: Three TMSG-1 overexpression clones were selected. Compared with the control cells, TMSG-1 overexpression MDA-MB-231 cells showed strong inhibition of proliferation and decreased clonogenicity in soft agar (P<0.05). Transfection of TMSG-1 into MDA-MB-231 cells significantly suppressed the cell invasion ability in vitro (decreased numbers of cells trespassing the matrigel in three experiments: 72.3+/-8.1, 85.0+/-4.2, and 73.5+/-7.8) in comparison with nave cells without transfection (187.5+/-2.1) and cells transfected with the control vector (162.3+/-6.8) (P<0.01). Transient transfection of TMSG-1 into MDA-MB-231 cells could promote cell apoptosis at 24 and 48 hours after transfection (P<0.05). CONCLUSIONS: TMSG-1 protein may have multiple functions in the regulation of proliferation, invasion and apoptosis of metastatic breast cancer cells, likely as a metastasis suppressor gene.