Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line.

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.

Reactive nitrogen and oxygen radicals formed during hepatic ischemia-reperfusion kill weakly metastatic colorectal cancer cells.

Microscopic infarcts develop within the livers of athymic nude mice during the first 24 h after human colorectal carcinoma (CRC) cells arrest within hepatic sinusoids. Because these regions are reperfused, essentially all weakly metastatic clone A and MIP-101 CRC cells die, whereas many highly metastatic CX-1 CRC cells survive. Because hepatic sinusoidal endothelial cells kill tumor cells in vitro by producing nitric oxide, superoxide anion, and other reactive oxygen and nitrogen species, our purpose was to determine whether reoxygenation of ischemic hepatic cultures in vitro forms toxic oxygen and nitrogen radicals that kill weakly but not highly metastatic CRC cells. CRC cells (10(7)) were labeled with rhodamine-dextran and calcein AM, cultured with cells from one mouse liver in a rotating suspension culture system for up to 24 h, and the metabolic activity of the CRC cells was determined. Liver fragments oxygenated normally before harvest were not toxic to either CRC cell line, but coculture with liver made ischemic by a 3-min ligation of the portal vein and hepatic artery in vivo before harvest and then cultured in oxygenated medium killed 50-70% of weakly metastatic clone A and MIP-101 cells at 24 h but <15% of highly metastatic CX-1 cells. Inhibition of nitric oxide synthase, addition of exogenous superoxide dismutase, but not catalase or hypoxia, during coculture blocked the killing of weakly metastatic CRC cells. Thus, reoxygenation of hepatic parenchymal and nonparenchymal cells after ischemia may form toxic species that eliminate weakly metastatic CRCs within 24 h of their arrest in the liver.