Exploring the robustness of DNA nanotubes framework for anticancer theranostics toward the 2D/3D clusters of hypopharyngeal respiratory tumor cells.

This study aimed to develop a robust approach for the early diagnosis and treatment of tumors. Short circular DNA nanotechnology synthesized a stiff and compact DNA nanotubes (DNA-NTs) framework. TW-37, a small molecular drug, was loaded into DNA-NTs for BH3-mimetic therapy to elevate the intracellular cytochrome-c levels in 2D/3D hypopharyngeal tumor (FaDu) cell clusters. After anti-EGFR functionalization, the DNA-NTs were tethered with a cytochrome-c binding aptamer, which can be applied to evaluate the elevated intracellular cytochrome-c levels via in situ hybridization (FISH) analysis and fluorescence resonance energy transfer (FRET). The results showed that DNA-NTs were enriched within the tumor cells via anti-EGFR targeting with a pH-responsive controlled release of TW-37. In this way, it initiated the triple inhibition of "BH3, Bcl-2, Bcl-xL, and Mcl-1". The triple inhibition of these proteins caused Bax/Bak oligomerization, leading to the perforation of the mitochondrial membrane. This led to the elevation of intracellular cytochrome-c levels, which reacted with the cytochrome-c binding aptamer to produce FRET signals. In this way, we successfully targeted 2D/3D clusters of FaDu tumor cells and achieved the tumor-specific and pH-triggered release of TW-37, causing tumor cell apoptosis. This pilot study suggests that anti-EGFR functionalized, TW-37 loaded, and cytochrome-c binding aptamer tethered DNA-NTs might be the hallmark for early tumor diagnosis and therapy.

Corrigendum to "The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors"[Journal of Pharmaceutical Analysis volume 11 (2021) 174-182].

[This corrects the article DOI: 10.1016/j.jpha.2020.10.003.].

Therapeutic interventions to urologic chronic pelvic pain syndrome and UPOINT system for clinical phenotyping: How far are we?

The assessment and management of urologic chronic pelvic pain syndrome (UCPPS), is controversial. It is classified by voiding symptoms, pelvic pain, and bladder pain, which is weekly treated, weekly understood, and bothersome. In the aspect of clinical efforts and research to help people with this syndrome have been hampered by the deficiency of a widely reliable, accepted, and a valuable tool to evaluate the patient symptoms and quality of life (QoL) impact. However, the etiology comes into sight is multifactorial, and available treatment options have been imprecise considerably in present years. We compiled the published literature on the assessment of the syndrome, a tentative role of pharmacological and non-pharmacological (conservative, alternative, and invasive therapy) interventions in eradicating the disease as well as improving symptoms. The previously published literature on animal models has established the association of immune systems in the etiology, pathogenesis, and progression of the disease. The UPOINT system for clinical phenotyping of UCPPS patients has six predefined domains that direct multimodal therapy, which would lead to significant symptom improvement in the medical field. The narrative review aims to scrutinize the fluctuating scientist's views on the evaluation of patient and multimodal treatment of the UPOINT system.

Drinking-water nitrate and cancer risk: A systematic review and meta-analysis.

BACKGROUND: Nitrate is an inorganic compound that occurs naturally in all surface and groundwater, although higher concentrations tend to occur only where fertilizers are used on the land. The regulatory limit for nitrate in public drinking water supplies was set to protect against infant methemoglobinemia, but other health effects were not considered. Risk of specific cancers and congenital disabilities may be increased when the nitrate is ingested, and nitrate is reduced to nitrite, which can react with amines and amides by nitrosation to form N-nitroso compounds which are known animal carcinogens. This study aims to evaluate the association between nitrate ingested through drinking water and the risk of developing cancers in humans. METHODS: We performed a systematic review following PRISMA and MOOSE guidelines. A literature search was performed using PubMed, EMBASE, the Cochrane Library databases, Web of Science and Google Scholars in the time-frame from their inception to January 2020, for potentially eligible publications. STATA version 12.0 was used to conduct meta-regression and a two-stage meta-analysis. RESULTS: A total of 48 articles with 13 different cancer sites were used for analysis. The meta-regression analysis showed stomach cancer had an association with the median dosage of nitrate from drinking water (t = 3.98, p = 0.0001, and adjusted R-squared = 50.61%), other types of cancers didn't show any association. The first stage of meta-analysis showed there was an association only between the risk of brain cancer & glioma (OR = 1.15, 95% CI: 1.06, 1.24) and colon cancer (OR = 1.11, 95% CI: 1.04, 1.17) and nitrate consumption in the analysis comparing the highest ORs versus the lowest. The 2nd stage showed there was an association only between the risk colon cancer (OR = 1.14, 95% CI: 1.04, 1.23) and nitrate consumption in the analysis comparing all combined higher ORs versus the lowest. CONCLUSION: This study showed that there is an association between the intake of nitrate from drinking water and a type of cancer in humans. The effective way of controlling nitrate concentrations in drinking water is the prevention of contamination (water pollution). Further research work on this topic is needed.

2D DNA nanoporous scaffold promotes osteogenic differentiation of pre-osteoblasts.

Biofunctional materials with nanomechanical parameters similar to bone tissue may promote the adherence, migration, proliferation, and differentiation of pre-osteoblasts. In this study, deoxyribonucleic acid (DNA) nanoporous scaffold (DNA-NPS) was synthesized by the polymerization of rectangular and double-crossover (DX) DNA tiles. The diagonally precise polymerization of nanometer-sized DNA tiles (A + B) through sticky end cohesion gave rise to a micrometer-sized porous giant-sheet material. The synthesized DNA-NPS exhibited a uniformly distributed porosity with a size of 25 ± 20 nm. The morphology, dimensions, sectional profiles, 2-dimensional (2D) layer height, texture, topology, pore size, and mechanical parameters of DNA-NPS have been characterized by atomic force microscopy (AFM). The size and zeta potential of DNA-NPS have been characterized by the zeta sizer. Cell biocompatibility, proliferation, and apoptosis have been evaluated by flow cytometry. The AFM results confirmed that the fabricated DNA-NPS was interconnected and uniformly porous, with a surface roughness of 0.125 ± 0.08035 nm. The elastic modulus of the DNA-NPS was 22.45 ± 8.65 GPa, which was comparable to that of native bone tissue. DNA-NPS facilitated pre-osteoblast adhesion, proliferation, and osteogenic differentiation. These findings indicated the potential of 2D DNA-NPS in promoting bone tissue regeneration.

A DNA-based nanocarrier for efficient cancer therapy.

The study aimed to achieve enhanced targeted cytotoxicity and cell-internalization of cisplatin-loaded deoxyribonucleic acid-nanothread (CPT-DNA-NT), mediated by scavenger receptors into HeLa cells. DNA-NT was developed with stiff-topology utilizing circular-scaffold to encapsulate CPT. Atomic force microscopy (AFM) characterization of the DNA-NT showed uniformity in the structure with a diameter of 50-150 nm and length of 300-600 nm. The successful fabrication of the DNA-NT was confirmed through native-polyacrylamide gel electrophoresis analysis, as large the molecular-weight (polymeric) DNA-NT did not split into constituting strands under applied current and voltage. The results of cell viability confirmed that blank DNA-NT had the least cytotoxicity at the highest concentration (512 nM) with a viability of 92% as evidence of its biocompatibility for drug delivery. MTT assay showed superior cytotoxicity of CPT-DNA-NT than that of the free CPT due to the depot release of CPT after DNA-NT internalization. The DNA-NT exhibited targeted cell internalizations with the controlled intracellular release of CPT (from DNA-NT), as illustrated in confocal images. Therefore, in vitro cytotoxicity assessment through flow cytometry showed enhanced apoptosis (72.7%) with CPT-DNA-NT (compared to free CPT; 64.4%). CPT-DNA-NT, being poly-anionic, showed enhanced endocytosis via scavenger receptors.

RETRACTED: Liposomal valinomycin mediated cellular K+ leak promoting apoptosis of liver cancer cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the corresponding author. It has been found that Fig 2B contains manipulated components, and Fig 5A partially overlaps with Fig 6 of a published paper authored by Mirza Muhammad Faran Ashraf Baig, et, al., The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors, J. Pharm. Anal. 11 (2021) 174-182, https://doi.org/10.1016/j.jpha.2020.10.003. The corresponding author indicated that they cannot guarantee the integrity of the images in the manuscript, as well as the conclusions of the paper. As a result, the Editor-in-Chief has decided to retract the paper. The corresponding author deeply regrets the circumstances and apologizes to the scientific community for not having detected this prior to publication.

The effective transfection of a low dose of negatively charged drug-loaded DNA-nanocarriers into cancer cells via scavenger receptors.

DNA-nanotechnology-based nano-architecture scaffolds based on circular strands were designed in the form of DNA-nanowires (DNA-NWs) as a polymer of DNA-triangles. Circularizing a scaffold strand (84-NT) was the critical step followed by annealing with various staple strands to make stiff DNA-triangles. Atomic force microcopy (AFM), native polyacrylamide gel electrophoresis (PAGE), UV-analysis, MTT-assay, flow cytometry, and confocal imaging were performed to assess the formulated DNA-NWs and cisplatin (CPT) loading. The AFM and confocal microscopy images revealed a uniform shape and size distribution of the DNA-NWs, with lengths ranging from 2 to 4 µm and diameters ranging from 150 to 300 nm. One sharp band at the top of the lane (500 bp level) with the loss of electrophoretic mobility during the PAGE (native) gel analysis revealed the successful fabrication of DNA-NWs. The loading efficiency of CPT ranged from 66.85% to 97.35%. MTT and flow cytometry results showed biocompatibility of the blank DNA-NWs even at 95% concentration compared with the CPT-loaded DNA-NWs. The CPT-loaded DNA-NWs exhibited enhanced apoptosis (22%) compared to the apoptosis (7%) induced by the blank DNA-NWs. The release of CPT from the DNA-NWs was sustained at < 75% for 6 h in the presence of serum, demonstrating suitability for systemic applications. The IC50 of CPT@DNA-NWs was reduced to 12.8 nM CPT, as compared with the free CPT solution exhibiting an IC50 of 51.2 nM. Confocal imaging revealed the targetability, surface binding, and slow internalization of the DNA-NWs in the scavenger-receptor-rich cancer cell line (HepG2) compared with the control cell line.

Therapeutic approach for global myocardial injury using bone marrow-derived mesenchymal stem cells by cardiac support device in rats.

Bone marrow-derived mesenchymal stem cells (BMSCs) have been considered a promising therapeutic approach to cardiovascular disease. This study intends to compare the effect of BMSCs through a standard active cardiac support device (ASD) and intravenous injection on global myocardial injury induced by isoproterenol. BMSCs were cultured in vitro, and the transplanted cells were labeled with a fluorescent dye CM-Dil. Isoproterenol (ISO) was injected into the rats; 2 weeks later, the labeled cells were transplanted into ISO-induced heart-jury rats through the tail vein or ASD device for 5 days. The rats were sacrificed on the first day, the third day, and the fifth day after transplantation to observe the distribution of cells in the myocardium by fluorescence microscopy. The hemodynamic indexes of the left ventricle were measured before sacrificing. H&E staining and Masson's trichrome staining were used to evaluate the cardiac histopathology. In the ASD groups, after 3 days of transplantation, there were a large number of BMSCs on the epicardial surface, and after 5 days of transplantation, BMSCs were widely distributed in the ventricular muscle. But in the intravenous injection group, there were no labeled-BMSCs distributed. In the ASD + BMSCs-three days treated group and ASD + BMSCs -five days-treated group, left ventricular systolic pressure (LVSP), the maximum rate of left ventricular pressure rise (+dP/dt), the maximum rate of left ventricular pressure decline (-dP/dt) increased compared with model group and intravenous injection group (P < 0.05). By giving BMSCs through ASD device, cells can rapidly and widely distribute in the myocardium and significantly improve heart function.

The integrin facilitated internalization of fibronectin-functionalized camptothecin-loaded DNA-nanofibers for high-efficiency anticancer effects.

Camptothecin (CMPT) in a free form is extremely cytotoxic as well as hydrophobic drug, and is considered to be highly contagious for systemic administration. The fibronectin (FN)-functionalized DNA-based nanocarrier has been designed to load CMPT and target integrin (αvß3) receptors which are highly expressed on the A549 cancer cells. Here, we report DNA nanocarrier in the form of DNA-nanofibers (DNA-NFs) capable of loading CMPT via strand intercalation in the GC (base pairs)-rich regions of the DNA duplex. Hence, our keen purpose was to explore the potential of DNA-NFs to load CMPT and assess the improvements of the outcomes in terms of enhanced therapeutic effects to integrin-rich A549 cancer cells with reduced cytotoxic effects to integrin-lacking HEK293 cells. DNA-NFs were formulated as a polymer of DNA triangles. DNA triangles arranged in a programmed way through the complementary overhangs present at the vertices. DNA triangles were primarily obtained through the annealing of the freshly circularized scaffold strands with the three distinct staple strands of specific sequences. The polymerized triangular tiles instead of forming two-dimensional nanosheets underwent self-coiling to give rise to DNA-NF-shaped structures. Flow cytometry and MTT assays were performed to observe cytotoxic and apoptotic effects on integrin-rich A549 cancer cells compared with the integrin-deficient HEK293 cells. AFM, native-page, and confocal experiments confirmed the polymerization of DNA triangles and the morphology of the resulting nanostructures. AFM and confocal images revealed the length of DNA-NFs to be 3-6 µm and the width from 70 to 110 nm. CMPT loading (via strands intercalation) in GC-rich regions of DNA-NFs and the FN functionalization (TAMRA tagged; red fluorescence) via amide chemistry using amino-modified strands of DNA-NFs were confirmed through the UV-shift analysis (> 10 nm shift) and confocal imaging. Blank DNA-NFs were found to be highly biocompatible in 2-640 µM concentrations. MTT assay and flow cytometry experiments revealed that CMPT-loaded DNA-NFs showed a dose-dependent decrease in the cell viability to integrin-rich A549 cancer cells compared with the integrin-deficient HEK293 cells. Conclusively, FN-functionalized, CMPT-loaded DNA-NFs effectively destroyed integrin-rich A549 cancer cells in a targeted manner compared with integrin-deficient HEK293 cells. Grapical abstract.

Therapeutic effects of Qianlie Tongli decoction on chronic prostatitis/chronic pelvic pain syndrome induced by peptide T2 in mice.

OBJECTIVES: This study was undertaken to reveal therapeutic effects and the preliminary mechanism of Chinese medicine formula Qianlie Tongli decoction (QTD) in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: A total of 50 male C57BL/6 mice were randomly divided into five groups. All groups except the control group were injected subcutaneously T2 peptide emulsion, which induced the CP/CPPS model. After the induction of CP/CPPS, the model group was given normal saline by oral gavage while low-dose, medium-dose and high-dose groups were treated with Chinese medicine formula. Micturition habits and pain behaviour of mice were analysed for each group. Haematoxylin and eosin (H&E) staining was used to investigate prostate inflammation. The serum level of tumour necrosis factor-α (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA) kit. KEY FINDINGS: Chinese medicine formula significantly reduced the number of urine spots and improved pain response frequency in the medium-dose and high-dose group. The high-dose group showed reduced considerably inflammatory lesion and inflammatory cell infiltration than the low-dose and medium-dose groups. Serum levels of TNF-α in the high-dose group were significantly reduced compared with the model group. CONCLUSIONS: The results demonstrated the therapeutic effects of Qianlie Tongli decoction in CP/CPPS mice by analysing clinically relevant symptoms (urinary tract system, pelvic pain and prostate inflammation) and preliminarily explored the inflammatory-related treatment mechanisms by measuring TNF-α.

Mass Transfer Modulation and Gas Mapping Based on Covalent Organic Frameworks-Covered Theta Micropipette.

Covalent organic frameworks (COFs) consist nanochannels that are fundamentally important for their application. Up to now, the effect of gas phase on COF nanochannels are hard to explore. Here, TAPB-PDA-COFs (triphenylbenzene-terephthaldehyde-COFs) was synthesized in situ at the tip of a theta micropipette. The COF-covered theta micropipette (CTP) create a stable gas-liquid interface inside the COF nanochannels, through which the humidity-modulated ion mass transfer in the COF nanochannels can be recorded by recording the current across the two channels of the theta micropipette. Results show that the humid air changes the mobility of the ions inside the COF nanochannels, which leads to the change of ionic current. Humid air showed different effects on the ion transfer depending on the solvent polarity index and vapor pressure. Current decreases linearly with the increase of relative humidity (RH) from 11% to 98%. The CTP was also mounted on the scanning electrochemical microscopy as a probe electrode for mapping micrometer-scale humidity distribution.

Synthesis of Ligand Functionalized ErbB-3 Targeted Novel DNA Nano-Threads Loaded with the Low Dose of Doxorubicin for Efficient In Vitro Evaluation of the Resistant Anti-Cancer Activity.

PURPOSE: Doxorubicin (Dox) being a hydrophobic drug needs a unique carrier for the effective encapsulation with uniformity in the aqueous dispersion, cell culture media and the biological-fluids that may efficiently target its release at the tumor site. METHODS: Circular DNA-nanotechnology was employed to synthesize DNA Nano-threads (DNA-NTs) by polymerization of triangular DNA-tiles. It involved circularizing a linear single-stranded scaffold strand to make sturdier and rigid triangles. DNA-NTs were characterized by the AFM and Native-PAGE tests. Dox binding and loading to the Neuregulin1 (NRG1) functionalized DNA based nano-threads (NF-DBNs) was estimated by the UV-shift analysis. The biocompatibility of the blank NRG-1/DNA-NTs and enhanced cytotoxicity of the NF-DBNs was assessed by the MTT assay. Cell proliferation/apoptosis was analyzed through the Flow-cytometry experiment. Cell-surface binding and the cell-internalization of the NF-DBNs was captured by the double-photon confocal microscopy (DPCM). RESULTS: The AFM images revealed uniform DNA-NTs with the diameter 30 to 80 nm and length 400 to 800 nm. PAGE native gel was used for the further confirmation of the successful assembly of the strands to synthesize DNA-NTs that gave one sharp band with the decreased electrophoretic mobility down the gel. MTT assay showed that blank DNA-NTs were biocompatible to the cells with less cytotoxicity even at elevated concentrations with most of the cells (94%) remaining alive compared to the dose-dependent enhanced cytotoxicity of NF-DBNs further evidenced by the Flow-cytometry analysis. CONCLUSION: Uniform and stiffer DNA-NTs for the potential applications in targeted drug delivery was achieved through circular DNA scaffolding.

Synthetic NRG-1 functionalized DNA nanospindels towards HER2/neu targets for in vitro anti-cancer activity assessment against breast cancer MCF-7 cells.

DNA based nano-carriers synthesized from short circular scaffolds (circular DNA nanotechnology) attains stiffer topology for ligand functionalization (neuregulin-1/NRG-1 ligand) and biological applications (targeted drug delivery). Daunorubicin (DR) is a hydrophobic chemical that requires robust vectors to efficiently encapsulate and avoid its free dispersion in water, biological media and cell culture. Here we design DNA nanospindels (DNA-NS) to efficiently load DR and target the (highly expressed) HER2/neu receptors on the plasma membrane of drug-resistant MCF-7 (breast cancer) cells. DNA-NS were synthesized by polymerizing the DNA-triangles (utilizing 84-nt short circular scaffold strand) into larger DNA nano-ribbons characterized by the native-PAGE testing. AFM results revealed the spinning of DNA nanoribbons on its (own) axis because of the intrinsic curvature of the DNA double helix resulting in the formation of the firm and twisted DNA-NS with the diameter (50-70 nm) and length (0.5-4 µm). DA loading onto DNA-NS was confirmed by the UV shift analysis. The MTT results with the blank DNA-NS evidenced its biocompatibility (remained value of 93%) compared to the decreased viability of the MCF-7 cells after treatment with DNA-NS (DR loaded). These findings were further supported by the analysis of cell proliferation/apoptosis through flow cytometry showing 64% apoptosis after treating with the DR loaded DNA-NS. Hence, through the short circular DNA nanotechnology, we have achieved a stiffer, uniform, and biocompatible DNA-NS for applications in the targeted therapy.

Chronic prostatitis/chronic pelvic pain syndrome and prostate cancer: study of immune cells and cytokines.

Prostate cancer and prostatitis are both significant health concerns. A large number of studies have established that the occurrence of the two is closely related. However, the most common prostatitis, type III chronic prostatitis/chronic pelvic pain syndromes (CP/CPPS), is reported to not correlate with the occurrence of prostate cancer. Although the etiology of CP/CPPS is unknown, it may be related to the autoimmune mechanism favored by most studies. Manipulating the immune system and targeting tumor microenvironment are promising new methods for the treatment of prostate cancer. Therefore, this review focuses on the immune cells and cytokines of CP/CPPS and prostate cancer from the perspective of biological immunology and immune microenvironment. We discuss T-regulatory (Treg) and T helper 17 (Th17) cells dysfunction, the abnormal regulation of T helper 1(Th1) and T helper 2 (Th2) cells, macrophages, and their related cytokines as key activators in CP/CPPS. In addition, we discuss the roles of Treg and Th17 cells, Th1 and Th2 cells, and related cytokines in modulating prostate cancer progression. This review highlights the concept that immune cells and cytokines provide a research strategy for the etiology of CP/CPPS and offer potentially promising targets for the treatment of prostate cancer.

Recent advances in the delivery of disulfiram: a critical analysis of promising approaches to improve its pharmacokinetic profile and anticancer efficacy.

BACKGROUND: Disulfiram (DSF) has a long history of being used as a first-line promising therapy for treatment of alcoholism in human. Besides its prominence in the treatment of alcoholism, extensive investigations have been carried out to explore other biomedical and pharmacological effects of DSF. Amongst other biomedical implications, plenty researches have shown evidence of promising anticancer efficacy of this agent for treatment of wide range of cancers such as breast cancer, liver cancer and lung carcinoma. METHODS: Electronic databases, including Google scholar, PubMed and Web of science were searched with the keywords disulfiram, nanoparticles, cancer, drug delivery systems. RESULT: Despite its excellent anticancer efficacy, the pharmaceutical significance and clinical applicability of DSF are hampered due to poor stability, low solubility, short plasma half-life, rapid metabolism, and early clearance from systemic circulation. Various attempts have been made to eradicate these issues. Nanotechnology based interventions have gained remarkable recognition in improving pharmacokinetic and pharmacodynamic profile of DSF by improving its stability and avoiding its degradation. CONCLUSION: The aim of the present review is to critically analyse all recent developments in designing various nanotechnology-based delivery systems, to ponder their relevance in improving stability, pharmacokinetic and pharmacodynamic profile, and achieving target-specific delivery of this agent to cancer cells to effectively eradicate cancer and abolish its metastasis. Nanotechnology is a novel approach for overcoming such obstacles faced presently, the results obtained so far using different novel drug delivery systems seem to be very promising to increase the stability and half-life of DSF. Graphical abstract Nanocrrier mediated drug delivery systems for disulfiram.

A DNA Nanodevice Simultaneously Activating the EGFR and Integrin for Enhancing Cytoskeletal Activity and Cancer Cell Treatment.

Cell-surface receptors (e.g., EGFR and integrin) and their interactions play determining roles in signal transduction and cytoskeletal activation, which affect cell attachment/detachment, invasion, motility, metastasis (intracellular), and cell-cell signaling. For instance, the interactions between the EGFR and integrin (α6ß4) may cause increased mechanical force and shear stress via enhanced cytoskeleton activation. Here, we design a DNA nanodevice (DNA-ND) that can simultaneously target the EGFR and integrin receptors on the caveolae. The piconewton (pN) forces in response to the EGFR-integrin coactivation can be sensed upon the unfolding of the DNA hairpin structure on the side arm of the device via changes of the fluorescence and plasmonic signals. We find that simultaneous activation of EGFR-integrin receptors causes enhanced signal transduction, contractions of the cells, and initiation of the biochemical pathways, thus resulting in a change of the cell division and endocytosis/exocytosis processes that affect the cell proliferation/apoptosis. The DNA-ND further enables us to visualize the cointernalization and degradation of the receptors by lysosomes, providing a novel approach toward bioimaging and mechano-pharmacology.

In-vitro Pre-Treatment of Cancer Cells with TGF-ß1: A Novel Approach of Tail Vein Lung Cancer Metastasis Mouse Model for Anti-Metastatic Studies.

BACKGROUND: Aggressive behavior of tumor metastasis comes from certain mutations, changes in cellular metabolic and signaling pathways that are majorly altered by tumor microenvironment (TME), its other components and growth factors like transforming growth factor-ß1 (TGF-ß1) which is chiefly known for its epithelial to mesenchymal transformation (EMT). EMT is a critical step of metastasis cascade in actual human lung cancer scenario. OBJECTIVE: Our present study is focused on unveiling the in-vivo metastatic behavior of TGF-ß1 treated lung cancer cells that undergo EMT. METHODS: The lung cancer epithelial A549 cells were treated in-vitro with TGF-ß1 (3-5ng/ml for 72 h) for EMT. After confirming the transformation of cells by phenotype modifications, wound healing and cell migration assay and qRT-PCR analyses of EMT biomarkers including E. Cadherin, Vimentin, Snail, Slug, MMP2 and MMP9; those TGF-ß1 modified cells were probed with fluorescent trackers and were injected into the tail vein of BALB/c nude mice for metastatic dissemination studies. RESULTS: Our findings indicate that the distribution of TGF-ß1 treated A549 cells as compared to W.T A549 towards lungs is less in terms of total relative fluorescent cluster count, however, the difference is insignificant (52±4, 60±5 respectively). Additionally, we show that TGF-ß1 treated cells tend to metastasize almost 2, 3, 1.5, 2 and 1.7 times more than W.T towards liver, brain, ovaries, bones and adrenal gland, respectively, which is very much like human lung cancer metastasis. CONCLUSION: Conclusively, it is the first study ever reporting that a pre-treatment of cells with TGF-ß1 for experimental lung cancer metastasis mouse model may portray a more precise approach for the development of potential therapeutic treatments. Additional pre-treatment studies with the application of other TME conditions like hypoxia and factors like NFκB, VEGF etc. may be a future prospect to develop a better understanding.