Curbing Breast Cancer by Altering V-ATPase Action on F-Actin, Heterochromatin, ETV7 and mTORC2 Signaling.

BACKGROUND/AIMS: Motivated by the vacuolar proton pump's importance in cancer, we investigate the effects of proton pump inhibition on breast cancer cell migration and proliferation, F-actin polymerization, lamin A/C, heterochromatin, and ETV7 expressions, nuclear size and shape, and AKT/mTOR signaling. METHODS: Lowly metastatic MCF7 and highly metastatic MDA-MB-231 breast cancer cells were treated with 120 nM of proton pump inhibitor Bafilomycin A1 for 24 hours. Cell migration was studied with wound- scratch assays, ATP levels with a chemiluminescent assay; cell proliferation was quantified by a cell area expansion assay. Nuclear size and shape were determined using DAPI nuclear stain and fluorescence microscopy. The levels of F-actin, lamin A/C, heterochromatin, and ETV7 were quantified using both immunocytochemistry and western blots; p-mTORC1, p-mTORC2, mTOR, p-AKT, and AKT were measured by western blots. RESULTS: We reveal that proton pump inhibition reduces F-actin polymerization, cell migration, proliferation, and increases heterochromatin in both lowly and highly metastatic cells. Surprisingly, Bafilomycin decreases lamin A/C in both cell lines. Inhibition has different effects on ETV7 expression in lowly and highly metastatic cells, as well as nuclear area, perimeter, and circularity. Bafilomycin also significantly decreases p-mTORC1, p-MTORC2, and MTOR expression in both cell lines, whereas it significantly decreases p-AKT in lowly metastatic cells and surprisingly significantly increases p-AKT in highly metastatic cells. Our proton pump inhibition protocol reduces V-ATPase levels (~25%) within three hours. V-ATPase levels vary in time for both control and inhibited cells, and inhibition reduces cellular ATP. CONCLUSION: Proton pumps promote F-actin polymerization and decrease heterochromatin, facilitating invasion. These pumps also upregulate both mTORC1 and mTORC2, thus highlighting the relevance of vacuolar proton pumps as metastatic cancer targets.

Modeling the change in European and US COVID-19 death rates.

Motivated by several possible differences in Covid-19 virus strains, age demographics, and face mask wearing between continents and countries, we focussed on changes in Covid death rates in 2020. We have extended our Covid-19 multicompartment model (Khan et al., 2020) to fit cumulative case and death data for 49 European countries and 52 US states and territories during the recent pandemic, and found that the case mortality rate had decreased by at least 80% in most of the US and at least 90% in most of Europe. We found that death rate decreases do not have strong correlations to other model parameters (such as contact rate) or other standard state/national metrics such as population density, GDP, and median age. Almost all the decreases occurred between mid-April and mid-June 2020, which corresponds to the time when many state and national lockdowns were relaxed resulting in surges of new cases. We examine here several plausible causes for this drop-improvements in treatment, face mask wearing, new virus strains, testing, potentially changing demographics of infected patients, and changes in data collection and reporting-but none of their effects are as significant as the death rate changes suggest. In conclusion, this work shows that a two death rate model is effective in quantifying the reported drop in death rates.

Parthenolide reverses the epithelial to mesenchymal transition process in breast cancer by targeting TGFbeta1: In vitro and in silico studies.

AIMS: Breast cancer metastasis is the leading cause of mortality among breast cancer patients. Epithelial to mesenchymal transition (EMT) is a biological process that plays a fundamental role in facilitating breast cancer metastasis. The present study assessed the efficacy of parthenolide (PTL Tanacetum parthenium) on EMT and its underlying mechanisms in both lowly metastatic, estrogen-receptor positive, MCF-7 cells and highly metastatic, triple-negative MDA-MB-231 cells. MAIN METHODS: MCF-7 and MDA-MB-231 cells were treated with PTL (2 µM and 5 µM). Cell viability was determined by MTT (3-(4,5-dimethy lthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was analyzed by the FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. The monolayer wound scratch assay was employed to evaluate cancer cell migration. Proteins were separated and identified by Western blotting. Gene expression was analyzed by quantitative real-time PCR. KEY FINDINGS: PTL treatment significantly reduced cell viability and migration while inducing apoptosis in both cell lines. Also, PTL treatment reverses the EMT process by decreasing the mesenchymal marker vimentin and increasing the epithelial marker E-cadherin compared to the control treatment. Importantly, PTL downregulates TWIST1 (a transcription factor and regulator of EMT) gene expression, concomitant with the reduction of transforming growth factor beta1 (TGFß1) protein and gene expression in both cell lines. Additionally, molecular docking studies suggest that PTL may induce anticancer properties by targeting TGFß1 in both breast cancer cell lines. SIGNIFICANCE: Our findings provide insights into the therapeutic potential of PTL to mitigate EMT and breast cancer metastasis. These promising results demand in vivo studies.

Shear Stress Increases V-H + -ATPase and Acidic Vesicle Number Density, and p-mTORC2 Activation in Prostate Cancer Cells.

INTRODUCTION: Cells in the tumor microenvironment experience mechanical stresses, such as compression generated by uncontrolled cell growth within a tissue, increased substrate stiffness due to tumor cell extracellular matrix (ECM) remodeling, and leaky angiogenic vessels which involve low fluid shear stress. With our hypothesis that shear stress increases V-H + -ATPase number density in prostate cancer cells via activation of the mTORC1 and mTORC2 pathways, we demonstrated and quantified such a mechanism in prostate cancer cells. METHODS: Moderately metastatic DU145 and highly metastatic PC3 prostate cancer cells were subjected to 0.05 dynes cm - 2 wall shear stress for 24 h, followed by immunocytochemistry and fluorescence measurements of ß 1 integrin, endosome, lysosome, V-H + -ATPase proton pump, mTORC1, and p-mTORC2 antibodies. Post shear stress migration assays, and the effects of vacuolar proton pump inhibitor Bafilomycin A1 (60 nM, 24 h) as well as shear stress on the ICC fluorescence intensity of the proteins of interest were conducted with DU145 cells. RESULTS: Low fluid shear stress increases the fluorescence intensity of ß 1 integrin, endosome, lysosome, V-H + -ATPase, mTORC1, and p-mTORC2 antibodies in PC3 and DU145 cells, and also increased cell migration. However, Bafilomycin A1 decreased fluorescence intensity of all of these proteins in DU145 cells exposed to shear stress, revealing that V-H + -ATPase controls the expression of these proteins. CONCLUSIONS: Prostate cancer cell mechanotransduction increases endosomes, lysosomes, and proton pumps-where increases have been associated with enhanced cancer aggressiveness. We also show that the prostate cancer cell's response to force promotes the cancer drivers mTORC1 and mTORC2.

Enhanced blebbing as a marker for metastatic prostate cancer.

Highly metastatic prostate cancer cells flowing through a microfluidic channel form plasma membrane blebs: they form 27% more than normal cells and have a lower stiffness (about 50%). Hypo-osmotic stress assays (with ∼ 50 % osmolarity) show 22% more blebbing of highly metastatic than moderately metastatic and 30% more than normal cells. Plasma membrane blebbing is known to provide important metastatic capabilities to cancer cells by aiding cell detachment from the primary tumor site and increasing cell deformability to promote cell migration through the extracellular matrix. Increased blebbing was attributed by others to decreased phosphorylated ezrin, radixin, and moesin (ERM) (p-ERM) protein expression-p-ERMs bind the plasma membrane to the actin cortex and reduced p-ERM expression can weaken membrane-cortex attachment. Myosin II also influences blebbing as myosin's natural contraction generates tension in the actin cortex. This increases cellular hydrostatic pressure, causes cortex rupture, cytoplasm flow out of the cortex, and hence blebbing. Highly metastatic cells are surprisingly found to express similar ezrin and myosin II levels but higher moesin levels in comparison with lowly metastatic or normal cells-suggesting that their levels, contrary to the literature [G. Charras and E. Paluch, Nat. Rev. Mol. Cell Biol. 9(9), 730-736 (2008); J.-Y. Tinevez, U. Schulze, G. Salbreux, J. Roensch, J.-F. Joanny, and E. Paluch, Proc. Natl. Acad. Sci. U.S.A. 106(44), 18581-18586 (2009); M. Bergert, S. D. Chandradoss, R. A. Desai, and E. Paluch, Proc. Natl. Acad. Sci. U.S.A. 109(36), 14434-14439 (2012); E. K. Paluch and E. Raz: Curr. Opin. Cell Biol. 25(5), 582-590 (2013)], are not important in metastatic prostate cell blebbing. Our results show that reduced F-actin is primarily responsible for increased blebbing in these metastatic cells. Blebbing can thus serve as a simple prognostic marker for the highly incident and lethal metastatic prostate cancer.

Aggressive prostate cancer cell nuclei have reduced stiffness.

It has been hypothesized that highly metastatic cancer cells have softer nuclei and hence would travel faster through confining environments. Our goal was to prove this untested hypothesis for prostate cells. Our nuclear creep experiments using a microfluidic channel with a narrow constriction show that stiffness of aggressive immortalized prostate cancer nuclei is significantly lower than that of immortalized normal cell nuclei and hence can be a convenient malignancy marker. Nuclear stiffness is found to be the highest for cells expressing high levels of lamin A/C but lowest for cells expressing low lamin A/C levels. Decreased chromatin condensation found in softer nuclei suggests that the former can also be a marker for aggressive cancers.

Flow-Induced Transport of Tumor Cells in a Microfluidic Capillary Network: Role of Friction and Repeated Deformation.

INTRODUCTION: Circulating tumor cells (CTCs) in microcirculation undergo significant deformation and frictional interactions within microcapillaries. To understand the physical parameters governing their flow-induced transport, we studied the pressure-driven flow of cancer cells in a microfluidic model of a capillary network. METHODS: Our microfluidic device contains an array of parallel constrictions separated by regions where cells can repetitively deform and relax. To characterize the transport behavior, we measured the entry time, transit time, and shape deformation of tumor cells as they squeeze through the network. RESULTS: We found that entry and transit times of cells are much lower after repetitive deformation as their elongated shape enables easy transport in subsequent constrictions. Furthermore, upon repetitive deformation, the cells were able to relieve only 25% of their 40% imposed compressional strain, suggesting that tumor cells might have undergone plastic deformation or fatigue. To investigate the influence of surface friction, we characterized the transport behavior in the absence and presence of bovine serum albumin (BSA) coating on the constriction walls. We observed that BSA coating reduces the entry and transit time significantly. Finally, using two breast tumor cell lines, we investigated the effect of metastatic potential on transport properties. We found that the cell lines could be distinguished only upon surface treatment with BSA, thus surface-induced friction is an indicator of metastatic potential. CONCLUSIONS: Our results suggest that pre-deformation can enhance the transport of CTCs in microcirculation and that frictional interactions with capillary walls can play an important role in influencing the transport of metastatic CTCs.

Blood plasma separation in a long two-phase plug flowing through disposable tubing.

We report a simple technique to separate plasma from blood in a flowing immiscible plug. We investigate the effect of various control parameters such as blood dilution, injection flow rate, observation time and fluid properties on plasma separation. We find that the technique works best for diluted blood samples at low plug velocities and long observation times. We postulate that the main mechanism responsible for efficient separation is the sedimentation of blood cells in the plug and their subsequent collection by the moving plug causing a significant accumulation of cells at the rear of the plug. We discuss the time scales determining the sedimentation, advection and collection of a blood cell in the immiscible plug and propose a phase diagram that is able to predict the operating space for effective plasma separation. We demonstrate that the technique allows for the extraction of more than 60% of the plasma by volume from 1 µL of diluted blood. We show the practical significance of this method by compartmentalizing the separated plasma into discrete microfluidic droplets and detecting cholesterol. This technique features low consumption of blood (nL-scale) and low shear rate (∼1 s(-1)). It is inexpensive, easy to use, and has the potential to be developed as an efficient point-of-care device for blood diagnostics in resource-poor environments. More advanced applications could also be envisioned by integrating our plasma separation method into existing microfluidic drop manipulation techniques.

Locomotion of C. elegans: a piecewise-harmonic curvature representation of nematode behavior.

Caenorhabditis elegans, a free-living soil nematode, displays a rich variety of body shapes and trajectories during its undulatory locomotion in complex environments. Here we show that the individual body postures and entire trails of C. elegans have a simple analytical description in curvature representation. Our model is based on the assumption that the curvature wave is generated in the head segment of the worm body and propagates backwards. We have found that a simple harmonic function for the curvature can capture multiple worm shapes during the undulatory movement. The worm body trajectories can be well represented in terms of piecewise sinusoidal curvature with abrupt changes in amplitude, wavevector, and phase.

Subdiffusive axial transport of granular materials in a long drum mixer.

Granular mixtures segregate radially by size when tumbled in a partially filled horizontal drum. The smaller component moves toward the axis of rotation and forms a buried core, which then splits into axial bands. Models have generally assumed that the axial segregation is opposed by diffusion. Using narrow pulses of the smaller component as initial conditions, we have characterized axial transport in the core. We find that the axial advance of the segregated core is well described by a self-similar concentration profile whose width scales as talpha, with alpha approximately 0.3<1/2. Thus, the process is subdiffusive rather than diffusive as previously assumed. We compare our results to two one-dimensional model equations which contain self-similarity and subdiffusion: a linear fractional diffusion model and the nonlinear porous medium equation.