Effects Of Long-Term Exposure To Phytochemicals and Nsaids On The Self-Renewal Of Leukemic Blast Progenitors In Vitro
Authors: Murohashi Ikuo , Miyahara Kazuma , Shimada Mai , Zhao Yan , Zhang Yi , Tanaka Ken-ichi , Hayashi Hiroyuki , Ihara Noriko 
Published: October 2019

Abstract

Objective
The preventive and therapeutic roles of non-steroid anti-inflammatory drugs (NSAIDs) and resveratrol (RSV) is well established. The preventive role of NSAIDs and aspirin, in particular, in colorectal cancer is well established. More recently, it has been suggested that aspirin may also have a therapeutic role. RSV, a polyphenol found in grape skins, has been proposed to reduce the risk of cancer development. Additionally, these studies indicate that resveratrol's chemoprevention effect is dose and duration dependent. The anti-cancer effects of ascorbic acid (vitamin C, Vit-C) have also been reported. However, the molecular mechanisms involved in NSAIDs- and these phytochemical-mediated tumor suppressing activities are not yet completely defined. We investigated the continuous additive effects of RSV, Vit-C, indomethacin (IND), and NS-398 on the growth of leukemic blast clonogenic cells in methylcellulose and the cumulative clonogenic cells in liquid suspension for up to one month by serial replating of leukemic cell lines. The interactive effects of additives were also investigated.

Methods
Myeloid (HL-60, K-562, MO7-E and U-937) and B lymphoid (Daudi, Raji and U-266) cell lines were used. RSV, Vit-C, IND, and NS-398 were added to the culture at a final concentration of 10, 300, 30, and 30μM, respectively. The concentrations of the additives in any of the cell lines were lower than IC50. Proportion of senescence was determined by using Senescence Detection Kit.

Results
Vit-C, RSV, IND and NS-398 significantly inhibited colony formation in three (43%), three (43%), three (43%) and four (57%) cell lines, respectively. In contrast, NS-398 significantly stimulated colony formation in U-937. Interactive effects between RSV/ Vit-C were synergistic (S) in four (57%), offset (O) in two (29%), and additive (A) in one (14%), those between RSV/IND were S in four (57%) and O in three (43%), and those between RSV/NS-398 were S in four (57%), O in two (29%) and A in one (14%), respectively. Logarithmic linear increase in the number of cumulative clonogenic cells in suspension was noted in any of the cell lines. RSV completely abrogated and partially but significantly inhibited the growth in three myeloid cell lines (MO7-E, U-937 and K-562) and four remaining lymphoid and myeloid cell lines, respectively. When RES was removed from the culture, complete recovery of the growth was noted. Vit-C, IND, and NS-398 only partially but significantly inhibited the growth in two (29%), one (14%) and one (14%) cell lines, respectively. In any of the cell lines, there was no correlation between the inhibitory effects of the additives on the clonogenic cell growth in methylcellulose and those in liquid suspension. RES enhanced p14 and/or p21 expression. Senescence was induced in all cell lines, except for Raji. RES induced significantly high proportion of senescence in MO7-E and K-562 after two-week culture comapred with control.

Conclusions
Phytochemicals and NSAIDs showed differential effects on colony formation in methylcellulose and cumulative clonogenic cell growth in suspension, probably owing to the different actions of these compounds in differentiation and self-renewal of these cell lines.

Disclosures:
No relevant conflicts of interest to declare.