Anti-invasive Effects Research Articles

Rhizopogon luteolus and Ganoderma adspersum Extracts Inhibit Invasion through the Crosstalk between Anti-oxidant Activity and Apoptosis Induced by pAKT/Rb.

Lung cancer is the primary cause of cancer-related deaths globally. Protein kinase B (AKT) protein is associated with many pathways in non-small cell lung cancer (NSCLC), such as proliferation, migration, invasion, and apoptosis. Mushrooms have a long history of being used in traditional medicine to treat various diseases. Scientists have been exploring the potential of mushrooms for their antioxidant and anticancer properties. In our study, the anti-oxidant, invasion, and apoptosis effects of mushroom extracts were investigated in NSCLC. Non-Small Cell Lung Cancer cell lines H1299, PC-3, and PC-14 were used in our study. After obtaining the extracts of Rhizopogon luteolus and Ganoderma adspersum, IC50 value was calculated as 25.04-11.73-16.54 ng/ul for R. luteolus and 2.97-1.53-1.01ug/ul for G. adspersum, respectively, in H1299, PC3 and PC14 cell lines. Afterward, proliferative and invasion effects, as well as apoptosis and anti-oxidant effects, were investigated using the IC50 dose. Western blotting was performed to investigate the pathways of these effects. According to the results of our study, Rhizopogon luteolus and Ganoderma adspersum extracts have anti-proliferative and anti-invasive effects on non-small lung cancer cell lines and induced apoptosis, which has been found to increase the anti-oxidant effect. It was found that this effect was due to cross-talk between antioxidant activity and the AKT-Rb pathway. We anticipate that Rhizopogon luteolus and Ganoderma adspersum extracts will be effective in cancer treatment by suppressing lung cancer cells via p-Akt and Rb.

The Proapoptotic Effect of MB-653 Is Associated with the Modulation of Metastasis and Invasiveness-Related Signalling Pathways in Human Colorectal Cancer Cells.

Colorectal cancer is one of the most common cancers worldwide and has a high mortality rate. In this study, we investigated the cytotoxic, proapoptotic, and anti-invasive effects of the synthetic indole phytoalexin MB-653. The antiproliferative effect was determined using an MTT assay, showing IC50 values of 5.8 ± 0.3 μmol/L for HCT116 cells and 6.1 ± 2.1 μmol/L for Caco2 cells. Flow cytometry and Western blot analysis were employed to investigate the molecular mechanisms underlying cytotoxicity, proapoptotic action, and anti-invasion effects. The proapoptotic activity was evidenced by the activation of caspases 3 and 7, mitochondrial dysfunction, and an increased number of apoptotic cells, confirmed by annexin V/PI and AO/PI staining. Additionally, MB-653 induces dose-dependent G2/M phase cell cycle arrest, the cause of which could be cyclin B1/CDC2 complex dysfunction and/or a decrease in α-tubulin protein expression. Another important observation was that MB-653 modulated several signalling pathways associated with various cellular activities, including survival, proliferation, tumour invasiveness, metastasis, and epithelial-mesenchymal transition (EMT). We further demonstrated its safety for topical and parenteral application. To sum up, our results indicate the real potential of MB-653 in treating colorectal cancer.

In-silico and In-vitro Evaluation of Novel Carboxamide Analogue on the Metastasis of Triple Negative Breast Cancer Cells Utilizing Novel PCPTC-loaded PEGylated-PLGA Nanocarriers.

This study aimed to determine the effects of novel N-{3-[(pyridin-4-yl)carbamoyl] phenyl} thiophene-2-carboxamide or PCPTC chemical moiety loaded Poly(lactic-co-glycolic acid)-Poly (Ethylene glycol) or (PLGA-PEGylated) NP as an anti-metastatic Ran GTPase therapeutic agent on MDA-MB231 triple-negative human breast cancer cells. Molecular docking and MD simulation was done to determine the binding potential of novel carboxamide PCPTC with Ran GTPase. PLGA and PLGA-PEG based NP encapsulating PCPTC were fabricated using the Modified Double Emulsion Solvent Evaporation Technique and characterized for size, zeta potential, polydispersity and morphology. In vitro evaluation of loaded nanoparticles such as cellular localization study, cell proliferation, cell migration, cell invasion and Ran Pull Down assay were carried out on MDA-MB231 breast cancer cells. Ran downregulation was determined by pull down assay. PCPTC with Ran GTPase exhibited strong structural stability based on in silico analysis. The average sizes of PCPTC loaded NP ranged between 166.3nm to 257.5nm and were all negatively charged. Scanning electron microscopy data showed that loaded NP were smooth and spherical. Fluorescence microscopy data confirmed the intracellular localization of loaded nanoparticles inside the MDA-MB231 cells. Cell proliferation assay (MTT assay) confirmed the cytotoxic effect of the loaded-NP when compared to blank nanoparticles. PCPTC-loaded NP inhibited metastasis and invasion of MDA-MB231 cells. This anti-metastatic and the anti-invasive effect was due to the Ran GTPase cycle blockage, which was confirmed by performing Ran Pull down assay. we propose that PCPTC is a promising compound to inhibit Ran GTPase and may act as a potential therapeutic agent against breast cancer. PCPTC-loaded NP successfully stopped the metastasis of MDA-MB231 cells by disrupting the Ran cycle.

Chlorogenic acid suppresses the expression of matrix metalloproteinase-7 and cell invasiveness to almost the same extent as isofraxidin in human colorectal cancer cells.

The expression of matrix metalloproteinase (MMP)-7 is reported to be correlated with invasion and metastasis of colorectal cancer (CRC). Therefore, the inhibition of MMP-7 would be beneficial for the suppression or prevention of CRC cell invasion and metastasis. The stem bark of Acanthopanax senticosus, a widely used medicinal herb, contains isofraxidin (IF) and chlorogenic acid (CGA) as major components. Previously we reported that IF suppressed the expression of MMP-7 and cell invasion in human hepatoma cells. In this study, we investigated the effects of CGA on cell invasion, MMP-7 mRNA expression and the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and compared it with those of IF in human CRC cells (HT-29). We found that CGA significantly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell invasion, MMP-7 expression and the expression of activated form of MMP-7 to almost the same extent as IF. Meanwhile, we also found that TPA-induced expression of MMP-7 mRNA and ERK1/2 phosphorylation were significantly suppressed when cells were exposed to the ERK1/2 inhibitor U0126 and that CGA was a little more potent than IF at inhibiting TPA-induced ERK1/2 phosphorylation. Taken together, the present results indicate that CGA suppresses cell invasion, MMP-7 expression and ERK1/2 phosphorylation to almost the same extent as IF and suggest that not only IF but also CGA suppresses cell invasion by inhibiting MMP-7 expression via the inhibition of at least ERK1/2 phosphorylation and Acanthopanax senticosus, which contains two components with anti-MMP-7 activity, may be a beneficial herb with anti-invasive effects against human CRC cells.

Asparagus officinalis L. extract exhibits anti-proliferative and anti-invasive effects in endometrial cancer cells and a transgenic mouse model of endometrial cancer.

Endometrial cancer is the most common malignancy of the female reproductive system in the United States. Asparagus officinalis is a versatile, nutrient-dense, low-calorie vegetable that contains various bioactive metabolites that have shown a variety of biologic functions beneficial to health. The metabolites from asparagus officinalis extracts or asparagus officinalis extracts exhibit significant anti-tumorigenic activity in some pre-clinical models of cancer. Endometrial cancer cells were used to study the effects of asparagus officinalis on anti-proliferation, anti-invasion and increased sensitivity to cisplatin, and obese and lean Lkb1 fl/fl p53 fl/fl mouse model of endometrial cancer was used to study the role of asparagus officinalis in tumor growth. Treatment with increasing concentrations of Asparagus officinalis extracts significantly inhibited cell proliferation, reduced glycolytic activity, induced cellular stress and apoptosis, caused cell cycle G1 arrest, increased the sensitivity of cells to cisplatin, reduced cell adhesion and invasion, and activation of AMPK and inhibition of the AKT/mTOR and MAPK signaling pathways in endometrial cancer cells. Moreover, asparagus officinalis extracts suppressed cell adhesion and invasion through the modulation of the epithelial-to-mesenchymal transition process. Asparagus officinalis extract treatment for 4weeks resulted in a significant reduction in tumor growth in Lkb1 fl/fl p53 fl/fl mice under both obese and lean conditions, with a decrease in Ki-67 and vascular endothelial growth factor expression and an increase in Bip expression in endometrial tumors. These findings provide strong preclinical evidence for the potential therapeutic benefit of asparagus officinalis extract as a novel dietary strategy in the treatment of endometrial cancer. Further clinical trials of dietary intervention of asparagus officinalis or combination with cisplatin in patients with endometrial cancer are warranted.

Mechanism of melanoma suppression by Prosopis juliflora derived alkaloids: Apoptosis induction and signaling pathway inhibition

BackgroundTraditional medicines remain integral to healthcare worldwide especially in developing countries. Our prior studies highlighted the anti-melanoma potential of Prosopis juliflora leave methanolic extract (PJLME), leading us to further isolate and characterize bioactive compounds from PJLME and evaluate their efficacy against melanoma. In this context, we identified alkaloid fractions derived from PJLME (PJLME-AF) as key agents in PJLME's cytotoxic effect against melanoma cells. PurposeThe purpose of this research was to evaluate the anti-cancer effects and mechanisms of PJLME-AF on melanoma cell lines. MethodsChemical Profiling of PJLME-AF was conducted by using LC-ESI-MS/MS. Cell viability and anti-migratory effects of PJLME-AF were assessed through MTT, wound healing and transwell assays. Mechanistic study of PJLME-AF was explored using ROS generation assay, apoptosis assays (Annexin V) assay, western blot (apoptotic protein, EMT markers, stem cell markers and signalling pathways), qPCR. In-vitro spheroid assay was performed to explored the PJLME-AF cytotoxic effects on tumour spheroid forming ability of melanoma cells. Additionally, the anti-tumor efficacy of PJLME-AF was tested in an in-vivo C57BL/6 mouse model. ResultsPJLME-AF demonstrated potent anti-proliferative, anti-migratory and anti-invasive effects on melanoma cells. Mechanistic study indicates that PJLME-AF strongly induce ROS generation and subsequent apoptotic cell death in melanoma cells. Furthermore, PJLME-AF downregulated epithelial-to-mesenchymal transition (EMT) proteins, stem cell markers and Akt/Erk cell survival proteins providing insights into its molecular mechanisms. Notably, PJLME-AF inhibited spheroid formation and reduced PD-L1 expression. Finally, anti-tumor activity of PJLME-AF was further confirmed in a in-vivo mice model. Additionally, PJLME-AF combined with dacarbazine, exhibited enhanced cytotoxicity on melanoma compared to the drug alone. ConclusionPJLME-AF exhibit promising natural anti-cancer agents for metastatic melanoma treatment. This research contributes to the development of novel, safe, and cost-effective agents for metastatic melanoma treatment and as a potential candidate for further cancer research.

Synthesis and Characterization of Thymol Carbon Nanodot Functionalized Silver Nanoparticles (ThCND-AgNPs) and Evaluation of Their Antiproliferative, Anti-Invasive, and Apoptotic Effects on OVCAR-3 Ovarian Cancer Cells.

Ovarian cancer belongs to the category of gynecological malignancies and unfortunately holds the distinction of being the most aggressive among them. It is ranked as the fifth highest cause of cancer-related deaths in women worldwide. The utilization of metal nanoparticles (NPs) linked with natural herbal molecules in biomedical applications has been on the rise. Thymol carbon nanodot functionalized silver nanoparticles (ThCND-AgNPs) were synthesized in an original manner and subjected to thorough characterization, including analysis of their size, morphology, and elemental composition. The aim of this study is to investigate the effects of the ThCND-AgNPs on cell proliferation, invasion, and apoptotic gene expressions in OVCAR-3 ovarian cancer cells. The effect of ThCND-AgNPs on cell viability in OVCAR cells was determined in a dose- and time-dependent manner using the XTT method. The effect on the expression changes of apoptotic-related genes was assessed through the Real-time PCR method, while the anti-invasive activity was measured using the matrigel invasion chamber assay. The ThCND-AgNP molecule exhibited a dose- and time-dependent reduction in cell proliferation in OVCAR-3 cells. The IC50 values were determined to be 388.53 μg/mL at 24 h and 145.683 μg/mL at 48 h. Furthermore, the molecule was found to reduce cell invasion by 51.12% compared with the control group in OVCAR-3 cells. In terms of apoptotic-related genes, Bcl-2 expression was downregulated, while BAX, CASPASE-3, -8, and -9 expressions were unregulated. In conclusion, the obtained data reveal the potential antiproliferative, apoptotic, and anti-invasive effects of our original ThCND-AgNP molecule in ovarian cancer. While these results need further confirmation through more detailed experiments, they will provide insights for future studies.

SNORA71A Downregulation Enhances Gemcitabine Sensitivity in Gallbladder Cancer Cells by Inducing Ferroptosis Through Inhibiting the AKT/NRF2/GPX4 Pathway.

Previous findings have indicated a marked upregulation of SNORA71A in gallbladder cancer (GBC) tissues compared to normal samples. However, the precise role and molecular mechanisms of SNORA71A in GBC remain largely unknown. Moreover, gemcitabine (GEM) drug resistance has been found to lead to unfavorable outcomes and recurrence in GBC patients. Therefore, this study aims to investigate the impact of SNORA71A on GBC and explore its potential effects on the sensitivity of GBC cells to GEM. RT-qPCR was conducted to assess SNORA71A level in matched normal and GBC tissues. Cell proliferation was examined through CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) assays. Additionally, the expression of proteins in GBC cells was analyzed using western blot assay. The level of SNORA71A was notably higher in GBC tissues relative to normal tissues. SNORA71A overexpression led to increased GBC cell proliferation and invasion. Conversely, SNORA71A deficiency strongly suppressed GBC cell proliferation and invasion and triggered cell apoptosis and ferroptosis. Additionally, downregulation of SNORA71A obviously enhanced the antiproliferative and anti-invasive effects of GEM on GBC cells, whereas these changes were reversed by inhibiting ferroptosis. Furthermore, deficiency of SNORA71A further augmented the GEM-induced downregulation of p-Akt, Nrf2, and GPX4 in NOZ cells; however, these effects were reversed by ferroptosis inhibition. Collectively, these findings suggested that downregulation of SNORA71A may increase the sensitivity of GBC cells to GEM by triggering ferroptosis through inhibiting the AKT/NRF2/GPX4 signaling pathway.

RETRACTION: A Multiplicity of Anti-Invasive Effects of Farnesyl Transferase Inhibitor SCH66336 in Human Head and Neck Cancer.

S. H. Oh , J.-H. Kang , J. K. Woo , O.-H. Lee , E. S. Kim , and H.-Y. Lee , "A Multiplicity of Anti-Invasive Effects of Farnesyl Transferase Inhibitor SCH66336 in Human Head and Neck Cancer," International Journal of Cancer 131, no. 3 (2012): 537-547, https://doi.org/10.1002/ijc.26373. The above article, published online on 16 August 2011 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor-in-Chief, Prof. Christoph Plass; the Union for International Cancer Control; and John Wiley & Sons Ltd. Following publication, concerns were raised by third parties regarding Figure 2D. Additional concerns were found during an investigation by the editorial office regarding Figures 1A and 4A. The authors were unable to provide comprehensive original data. The retraction has been agreed because parts of the figures and the underlying data were duplicated. The editors no longer have confidence in the data reported in this article or in the interpretation of the results presented. The authors S. H. O, J.-H. K., and H.-Y. L. were informed of the decision to retract; J. K. W., O.-H. L., and E. S. K. could not be contacted.

An Anti-Invasive Role for Mdmx through the RhoA GTPase under the Control of the NEDD8 Pathway.

Mdmx (Mdm4) is established as an oncogene mainly through repression of the p53 tumour suppressor. On the other hand, anti-oncogenic functions for Mdmx have also been proposed, but the underlying regulatory pathways remain unknown. Investigations into the effect of inhibitors for the NEDD8 pathway in p53 activation, human cell morphology, and in cell motility during gastrulation in Xenopus embryos revealed an anti-invasive function of Mdmx. Through stabilisation and activation of the RhoA GTPase, Mdmx is required for the anti-invasive effects of NEDDylation inhibitors. Mechanistically, through its Zn finger domain, Mdmx preferentially interacts with the inactive GDP-form of RhoA. This protects RhoA from degradation and allows for RhoA targeting to the plasma membrane for its subsequent activation. The effect is transient, as prolonged NEDDylation inhibition targets Mdmx for degradation, which subsequently leads to RhoA destabilisation. Surprisingly, Mdmx degradation requires non-NEDDylated (inactive) Culin4A and the Mdm2 E3-ligase. This study reveals that Mdmx can control cell invasion through RhoA stabilisation/activation, which is potentially linked to the reported anti-oncogenic functions of Mdmx. As inhibitors of the NEDD8 pathway are in clinical trials, the status of Mdmx may be a critical determinant for the anti-tumour effects of these inhibitors.

Development of Novel ROCK Inhibitors via 3D-QSAR and Molecular Docking Studies: A Framework for Multi-Target Drug Design.

Background/Objectives: Alterations in the actin cytoskeleton correlates to tumor progression and affect critical cellular processes such as adhesion, migration and invasion. Rho-associated coiled-coil-containing protein kinases (ROCK1 and ROCK2), important regulators of the actin cytoskeleton, are frequently overexpressed in various malignancies. The aim of this study was therefore to identify the key structural features of ROCK1/ROCK2 inhibitors using computer-aided drug design (CADD) approaches. In addition, new developed ROCK inhibitors provided a significant framework for the development of multitarget therapeutics-ROCK/HDAC (histone deacetylases) multitarget inhibitors. Methods: 3D-QSAR (Quantitative structure-activity relationship study) and molecular docking study were employed in order to identify key structural features that positively correlate with ROCK inhibition. MDA-MB-231, HCC1937, Panc-1 and Mia PaCa-2 cells were used for evaluation of anticancer properties of synthesized compounds. Results: C-19 showed potent anti-cancer properties, especially enhancement of apoptosis and cell cycle modulation in pancreatic cancer cell lines. In addition, C-19 and C-22 showed potent anti-migratory and anti-invasive effects comparable to the well-known ROCK inhibitor fasudil. Conclusions: In light of the results of this study, we propose a novel multi-target approach focusing on developing dual HDAC/ROCK inhibitors based on the structure of both C-19 and C-22, exploiting the synergistic potential of these two signaling pathways to improve therapeutic efficacy in metastatic tumors. Our results emphasize the potential of multi-target ROCK inhibitors as a basis for future cancer therapies.

Alpha lipoic acid diminishes migration and invasion in hepatocellular carcinoma cells through an AMPK-p53 axis

Hepatocellular carcinoma (HCC) associated with viral or metabolic liver diseases is a growing cancer without effective therapy. AMPK is downregulated in HCC and its activation diminishes tumor growth. Alpha lipoic acid (ALA), an indirect AMPK activator that inhibits hepatic steatosis, shows antitumor effects in different cancers. We aimed to study its putative action in liver-cancer derived cell lines through AMPK signaling. We performed cytometric studies for apoptosis and cell cycle, and 2D and 3D migration analysis in HepG2/C3A and Hep3B cells. ALA led to significant inhibition of cell migration/invasion only in HepG2/C3A cells. We showed that these effects depended on AMPK, and ALA also increased the levels and nuclear compartmentalization of the AMPK target p53. The anti-invasive effect of ALA was abrogated in stable-silenced (shTP53) versus isogenic-TP53 HepG2/C3A cells. Furthermore, ALA inhibited epithelial-mesenchymal transition (EMT) in control HepG2/C3A but not in shTP53 nor in Hep3B cells. Besides, we spotted that in patients from the HCC-TCGA dataset some EMT genes showed different expression patterns or survival depending on TP53. ALA emerges as a potent activator of AMPK-p53 axis in HCC cells, and it decreases migration/invasion by reducing EMT which could mitigate the disease in wild-type TP53 patients.

Sulindac exhibits anti-proliferative and anti-invasive effects in uterine serous carcinoma cells

PurposeUterine serous carcinoma (USC) is a highly aggressive and frequently recurring subtype of endometrial cancer with limited treatment options for advanced or recurrent stages. Sulindac, a classic non-steroidal anti-inflammatory drug, has demonstrated anti-tumor activity in several pre-clinical tumor models. This study aims to evaluate the effect of sulindac on cell proliferation and invasion in USC cells.MethodsHuman USC cell lines ARK-1 and SPEC2 were treated with different concentrations of sulindac. Cell proliferation was assessed using MTT and colony formation assays. ELISA assays measured cellular stress, cleaved caspase 3 activity, antioxidant ability, and adhesion. Cell cycle arrest was evaluated by Cellometer. The invasive capability was detected by wound healing assay. Western blotting was used to analyze the changes in protein expression induced by sulindac.ResultsExposure to sulindac decreased cellular viability in a dose-dependent manner in ARK-1 and SPEC2 cells. Sulindac effectively inhibited cell cycle progression, increased cellular stress, caused apoptosis, and reduced cell adhesion and invasion in USC cells. Additionally, sulindac decreased the expression of COX-2 and blocked phosphorylation of NF-κB induced by TNF-α.ConclusionSulindac is a potential therapeutic agent for USC that deserves further exploration in pre-clinical studies and potentially future clinical trials.

NSC-38270 Exhibits Anti-invasive and Pro-apoptotic Effects on Hepatocellular Carcinoma Huh7 Cells.

Hepatocellular carcinoma (HCC) is a main type of liver cancer with high metastatic potential, and its incidence is steadily increasing worldwide. However, the development of new drugs for the treatment of HCC is still insufficient. This study aimed to determine the anticancer effect of NSC-38270, a natural product, on HCC. After treating HCC Huh7 cells with NSC-38270, cell growth, wound healing, migration, and invasion assays were conducted. We investigated the effects of NSC-38270 on Twist1, a crucial epithelial-mesenchymal transition (EMT)-related transcription factor. In addition, apoptosis, histone H2A.X activation, and cell morphology assays were performed in Huh7 and immortalized normal liver cells following treatment with NSC-38270. NSC-38270 reduced the migration and invasion ability of Huh7 cells, accompanied by a decrease in Twist1. Furthermore, NSC-38270 induced apoptosis in Huh7 cells, whereas apoptosis was not observed in immortalized normal liver cells (THLE-2 cells and Chang liver cells). NSC-38270 exhibited significant inhibitory effects on the migration and invasion of Huh7 cells by repressing Twist1. Importantly, it induced cancer cell-specific apoptotic effects. These findings suggest that NSC-38270 holds promising potential as a therapeutic candidate for cancer treatment.

Antitumoral and Antimetastatic Activity by Mixed Chelate Copper(II) Compounds (Casiopeínas®) on Triple-Negative Breast Cancer, In Vitro and In Vivo Models.

Triple-negative breast cancer (TNBC), accounting for 15-20% of all breast cancers, has one of the poorest prognoses and survival rates. Metastasis, a critical process in cancer progression, causes most cancer-related deaths, underscoring the need for alternative therapeutic approaches. This study explores the anti-migratory, anti-invasive, anti-tumoral, and antimetastatic effects of copper coordination compounds Casiopeína IIIia (CasIIIia) and Casiopeína IIgly (CasIIgly) on MDA-MB-231 and 4T1 breast carcinoma cell lines in vitro and in vivo. These emerging anticancer agents, mixed chelate copper(II) compounds, induce apoptosis by generating reactive oxygen species (ROS) and causing DNA damage. Whole-transcriptome analysis via gene expression arrays indicated that subtoxic concentrations of CasIIIia upregulate genes involved in metal response mechanisms. Casiopeínas® reduced TNBC cell viability dose-dependently and more efficiently than Cisplatin. At subtoxic concentrations (IC20), they inhibited random and chemotactic migration of MDA-MB-231 and 4T1 cells by 50-60%, similar to Cisplatin, as confirmed by transcriptome analysis. In vivo, CasIIIia and Cisplatin significantly reduced tumor growth, volume, and weight in a syngeneic breast cancer model with 4T1 cells. Furthermore, both compounds significantly decreased metastatic foci in treated mice compared to controls. Thus, CasIIIia and CasIIgly are promising chemotherapeutic candidates against TNBC.

An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout.

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

Exploring the potential of tocopherols: mechanisms of action and perspectives in the prevention and treatment of breast cancer.

Currently, breast cancer is the most common cause of mortalitycaused by neoplasia in women worldwide. The unmet challenges of conventional cancer therapy are chemoresistance and lack of selectivity, which can lead to serious side effects in patients; therefore,new treatments based on natural compounds that serve as adjuvants in breast cancer therapyare urgently needed. Tocopherols are naturally occurring antioxidant compounds that have shown antitumor activity against several types of cancer, including breast cancer. This review summarizes the antitumoral activity of tocopherols, such as theantiproliferative, apoptotic, anti-invasive, and antioxidant effects of tocopherols, through different molecular mechanisms. According to the studies described, α-T, δ-T and γ-T are the most studied in breast tumor cells; however, α-T and γ-T show a more critical antitumor activity and significant potential as a complements to chemotherapeutic drugs against breast cancer, enhancing toxicity against tumor cells and preventing cytotoxicity in nontumor cells. However, the possible relationship between tocopherol intake, related to concentration,and the promotion of cancer in particular cases should not be ruled out, so additional studies are required to determine the correct dose to obtain the desired antitumor effect. Moreover, nanomicelles of D-α-tocopherol have promising potential as pharmaceutical excipients for drug delivery to improve the cytotoxicity and selectivity of first-line chemotherapeutics against breast cancer.

The Discovery of GIT1/β-Pix Inhibitors: Virtual Screening and Biological Evaluation of New Small-molecule Compounds with Anti-invasion Effect in Gastrointestinal Neoplasms.

GIT1 (G-protein-coupled receptor kinase interacting protein-1) has been found to be highly related with cancer cell invasion and metastasis in many cancer types. β-Pix (p21-activated kinase-interacting exchange factor) is one of the proteins that interact with GIT1. Targeting GIT1/β-Pix complex might be a potential therapeutic strategy for interfering cancer metastasis. However, at present, no well-recognized small-molecule inhibitor targeting GIT1/β-Pix is available. Thus, we aim to discover novel GIT1/β-Pix inhibitors with simple scaffold, high activity and low toxicity to develop new therapeutic strategies to restrain cancer metastasis. GIT1/β-Pix inhibitors were identified from ChemBridge by virtual screening. Briefly, the modeling of GIT1 was performed and the establishment of GIT1/β-Pix binding pocket enabled the virtual screening to identify the inhibitor. In addition, direct binding of the candidate molecules to GIT1 was detected by biolayer interferometry (BLI) to discover the hit compound. Furthermore, the inhibitory effect on invasion of stomach and colon cancer cells in vitro was carried out by the transwell assay and detection of epithelial-mesenchymal transition (EMT)-related proteins. Finally, the binding mode of hit compound to GIT1 was estimated by molecular dynamics simulation to analyze the key amino residues to guide further optimization. We selected the top 50 compounds from the ChemBridge library by virtual screening. Then, by skeleton similarity analysis nine compounds were selected for further study. Furthermore, the direct interaction of nine compounds to GIT1 was detected by BLI to obtain the best affinitive compound. Finally, 17302836 was successfully identified (KD = 84.1±2.0 μM). In vitro tests on 17302836 showed significant anti-invasion effect on gastric cancer and colorectal cancer. We discovered a new GIT1/β-Pix inhibitor (17302836) against gastrointestinal cancer invasion and metastasis. This study provides a promising candidate for developing new GIT1/β-Pix inhibitors for tumor treatment.

Monastrol suppresses invasion and metastasis in human colorectal cancer cells by targeting fascin independent of kinesin-Eg5 pathway

Rearrangement of the actin cytoskeleton is a prerequisite for carcinoma cells to develop cellular protrusions, which are required for migration, invasion, and metastasis. Fascin is a key protein involved in actin bundling and is expressed in aggressive and invasive carcinomas. Additionally, fascin appears to be involved in tubulin-binding and microtubule rearrangement. Pharmacophoric-based in silico screening was performed to identify compounds with better fascin inhibitory properties than migrastatin, a gold-standard fascin inhibitor. We hypothesized that monastrol displays anti-migratory and anti-invasive properties via fascin blocking in colorectal cancer cell lines. Biophysical (thermofluor and ligand titration followed by fluorescence spectroscopy), biochemical (NMR), and cellular assays (MTT, invasion of human tissue), as well as animal model studies (zebrafish invasion) were performed to characterize the inhibitory effect of monastrol on fascin activity. In silico analysis revealed that monastrol is a potential fascin-binding compound. Biophysical and biochemical assays demonstrated that monastrol binds to fascin and interferes with its actin-bundling activity. Cell culture studies, including a 3D human myoma disc model, showed that monastrol inhibited fascin-driven cytoplasmic protrusions as well as invasion. In silico, confocal microscopy, and immunoprecipitation assays demonstrated that monastrol disrupted fascin-tubulin interactions. These anti-invasive effects were confirmed in vivo. In silico confocal microscopy and immunoprecipitation assays were carried out to test whether monastrol disrupted the fascin-tubulin interaction. This study reports, for the first time, the in vitro and in vivo anti-invasive properties of monastrol in colorectal tumor cells. The number and types of interactions suggest potential binding of monastrol across actin and tubulin sites on fascin, which could be valuable for the development of antitumor therapies.

Estetrol/GPER/SERPINB2 transduction signaling inhibits the motility of triple-negative breast cancer cells

BackgroundEstetrol (E4) is a natural estrogen produced by the fetal liver during pregnancy. Due to its favorable safety profile, E4 was recently approved as estrogenic component of a new combined oral contraceptive. E4 is a selective ligand of estrogen receptor (ER)α and ERβ, but its binding to the G Protein-Coupled Estrogen Receptor (GPER) has not been described to date. Therefore, we aimed to explore E4 action in GPER-positive Triple-Negative Breast Cancer (TNBC) cells.MethodsThe potential interaction between E4 and GPER was investigated by molecular modeling and binding assays. The whole transcriptomic modulation triggered by E4 in TNBC cells via GPER was explored through high-throughput RNA sequencing analyses. Gene and protein expression evaluations as well as migration and invasion assays allowed us to explore the involvement of the GPER-mediated induction of the plasminogen activator inhibitor type 2 (SERPINB2) in the biological responses triggered by E4 in TNBC cells. Furthermore, bioinformatics analysis was aimed at recognizing the biological significance of SERPINB2 in ER-negative breast cancer patients.ResultsAfter the molecular characterization of the E4 binding capacity to GPER, RNA-seq analysis revealed that the plasminogen activator inhibitor type 2 (SERPINB2) is one of the most up-regulated genes by E4 in a GPER-dependent manner. Worthy, we demonstrated that the GPER-mediated increase of SERPINB2 is engaged in the anti-migratory and anti-invasive effects elicited by E4 in TNBC cells. In accordance with these findings, a correlation between SERPINB2 levels and a good clinical outcome was found in ER-negative breast cancer patients.ConclusionsOverall, our results provide new insights into the mechanisms through which E4 can halt migratory and invasive features of TNBC cells.