The ROCK-II isoform of Rho's downstream effector, Rho kinase, has been linked with greater invasion and metastasis in solid tumors. We have previously shown that ROCK-II is overexpressed at the advancing edge of colon cancers. The mechanism whereby ROCK-II contributes invasion, particularly in the setting of colon cancer, remains to be elucidated fully. To better understand its contribution, we evaluated ROCK-II expression in both non-malignant (NCM460 and IEC-6) and malignant (Caco-2 E, SW620, and HCT-116) intestinal epithelial cell lines grown in type I collagen scaffolds. Using multiphoton microscopy, we observed that ROCK-II localized to the actin cytoskeleton in non-malignant cells but localized to the cell periphery as focal collections with an absence of adjacent collagen in all colon cancer cell lines. By transmission electron microscopy, these collections corresponded with finger-like projections previously described as invadopodia. Immunogold staining with cortactin, matrix metalloprotease (MMP)-2, -9, and -13 confirmed that these were indeed invadopodia. To further link ROCK-II to colon cancer invasion, we treated non-malignant and malignant intestinal epithelial cell lines with ROCK-II siRNA and evaluated depth of invasion, proliferation, and MMP-2, -9, and -13 activities. The most striking effect was seen in the highly tumorigenic cell lines, SW620 and HCT-116, wherein ROCK-II knockdown resulted in a two-fold or more reduction in invasion. This reduction in invasion was not due to a decrease in cell proliferation, as a significant reduction in proliferation was only observed in the two non-malignant intestinal cell lines. Finally, both MMP-2 and -13 activities were significantly decreased in all colon cancer cell lines. Taken together, these data suggest for the first time that ROCK-II is a critical mediator of colon cancer cell invasion through its modulation of MMP-2 and -13 at the site of invadopodia but regulates proliferation in non-malignant intestinal cells.

The concept of using tissue density as a mechanism to diagnose a tumor has been around for centuries. However, this concept has not been sufficiently explored in a laboratory setting. Therefore, in this paper, we observed the effects of cell density and extracellular matrix (ECM) density on colon cancer invasion and proliferation using SW620 cells. We also attempted to inhibit ROCK-I to determine its effect on cell invasion and proliferation using standard molecular biology techniques and advanced imaging. Increasing cell seeding density resulted in a 2-fold increase in cell invasion as well as cell proliferation independent of treatment with Y-27632. Increasing collagen I scaffold density resulted in a 2.5-fold increase in cell proliferation while treatment with Y-27632 attenuated this effect although 1.5 fold increase in cell invasion was observed in ROCK inhibited samples. Intriguingly, ROCK inhibition also resulted in a 3.5-fold increase in cell invasion within 3D collagen scaffolds for cells seeded at lower densities. We show in this paper that ROCK-I inhibition leads to increased invasion within 3D collagen I microenvironments. This data suggests that although ROCK inhibitors have been used clinically to treat several medical conditions, its effect largely depends on the surrounding microenvironment.

Background: Meditation is gaining recognition as a tool to impact health and well-being. Samyama is an 8-day intensive residential meditation experience conducted by Isha Foundation requiring several months of extensive preparation and vegan diet. The health effects of Samyama have not been previously studied. The objective was to assess physical and emotional well-being before and after Samyama participation by evaluating psychological surveys and objective health biomarkers.Methods: This was an observational study of 632 adults before and after the Isha Samyama retreat. All participants were invited to complete surveys. Controls included household significant others. Surveys were completed at baseline (T1), just before Samyama (T2), immediately after Samyama (T3), and 3 months later (T4) to assess anxiety, depression, mindfulness, joy, vitality, and resilience through validated psychometric scales. Voluntary blood sampling for biomarker analysis was done to assess hemoglobin (Hb), HbA1c, lipid profile, and C-reactive protein (CRP). Primary outcomes were changes in psychometric scores, body weight, and blood biomarkers.Results: Depression and anxiety scores decreased from T1 to T3, with the effect most pronounced in participants with baseline depression or anxiety. Scores at T4 remained below baseline for those with pre-existing depression or anxiety. Vitality, resilience, joy, and mindfulness increased from T1 to T3 (sustained at T4). Body weight decreased by 3% from T1 to T3. Triglycerides (TG) were lower from T2 to T3. Participants had lower HbA1c and HDL at T2, and lower CRP at all timepoints compared with controls.Conclusions: Participation in the Isha Samyama program led to multiple benefits. The 2-month preparation reduced anxiety, and participants maintained lower anxiety levels at 3 months post-retreat. Physical health improved over the course of the program as evidenced by weight loss and improved HbA1C and lipid profile. Practices associated with the Samyama preparation phase and the retreat may serve as an effective way to improve physical and mental health. Future studies may examine their use as an alternative therapy in patients with depression and/or anxiety.Clinical Trial Registration:www.ClinicalTrials.gov, Identifier: 1801728792. Registered retrospectively on 4/17/2020.

Background. Anxiety and depression are common in the modern world, and there is growing demand for alternative therapies such as meditation. Meditation can decrease perceived stress and increase general well-being, although the physiological mechanism is not well-characterized. Endocannabinoids (eCBs), lipid mediators associated with enhanced mood and reduced anxiety/depression, have not been previously studied as biomarkers of meditation effects. Our aim was to assess biomarkers (eCBs and brain-derived neurotrophic factor [BDNF]) and psychological parameters after a meditation retreat. Methods. This was an observational pilot study of adults before and after the 4-day Isha Yoga Bhava Spandana Program retreat. Participants completed online surveys (before and after retreat, and 1 month later) to assess anxiety, depression, focus, well-being, and happiness through validated psychological scales. Voluntary blood sampling for biomarker studies was done before and within a day after the retreat. The biomarkers anandamide, 2-arachidonoylglycerol (2-AG), 1-arachidonoylglycerol (1-AG), docosatetraenoylethanolamide (DEA), oleoylethanolamide (OLA), and BDNF were evaluated. Primary outcomes were changes in psychological scales, as well as changes in eCBs and BDNF. Results. Depression and anxiety scores decreased while focus, happiness, and positive well-being scores increased immediately after retreat from their baseline values (P<0.001). All improvements were sustained 1 month after BSP. All major eCBs including anandamide, 2-AG, 1-AG, DEA, and BDNF increased after meditation by > 70% (P<0.001). Increases of ≥20% in anandamide, 2-AG, 1-AG, and total AG levels after meditation from the baseline had weak correlations with changes in happiness and well-being. Conclusions. A short meditation experience improved focus, happiness, and positive well-being and reduced depression and anxiety in participants for at least 1 month. Participants had increased blood eCBs and BDNF, suggesting a role for these biomarkers in the underlying mechanism of meditation. Meditation is a simple, organic, and effective way to improve well-being and reduce depression and anxiety.

Polyethylene glycol (PEG) has been previously shown to protect against enteric pathogens and prevent colon cancer invasion. To determine if PEG could indeed protect against previously observed pro-invasive effects of commensal E. coli and EPEC, Caco-2 cells grown in an in vitro model of colon cancer were infected with strains of human commensal E. coli or EPEC and treated with 10% PEG 3350, PEG 8000, and PEG 20,000, respectively. At 24 hours after infection, MMP-1 and MMP-13 activities, cell cluster thickness, depth of invasion, and proliferation were determined using standard molecular biology techniques and advanced imaging. We found that higher molecular weight PEG, especially PEG 8000 and 20,000, regardless of bacterial infection, increased proliferation and depth of invasion although a decrease in cellular density and MMP-1 activity was also noted. Maximum proliferation and depth of invasion of Caco-2 cells was observed in scaffolds treated with a combination of commensal E. coli strain, HS4 and PEG 8000. In conclusion, we found that PEG 8000 increased cell proliferation and led to the preservation of cell density in cells treated with commensal bacteria. This is important, because the preservation of a proliferative response in colon cancer results in a more chemo-responsive tumor.