Evaluation of a Synergistic Effect of L-Arginine on the Anticancer Activity of Doxorubicin by Using a Co-culture System

Abstract

In the early stage of tumor development, tumor associated macrophage (TAM) works to suppress tumor growth by secreting soluble factors including nitric oxide (NO). L-Arginine (Arg) is a substrate of nitric oxide synthase (NOS) expressed in TAM. Here we examined whether NO produced from Arg by macrophages works to enhance the effect of the anti-cancer drug, doxorubicin (Dox) by using co-culture system of cancer cells with macrophages. By employing colorimetric analyses methods (Griess Reagent and Cell Counting kit-8), we found that NO produced from Arg by co-cultured macrophages could enhance the cytotoxic effect of Dox to cancer cells. Moreover, we found that the augmentation is affected by the order of the addition of Arg and Dox. Prior addition of Arg to Dox and simultaneous addition showed the same enhancement effect, but prior addition of Dox to Arg abolished the augmentation. This suggests that the co-administration of Arg with Dox would be effective treatment to improve chemo-therapies.

Triggering the nanophase separation of albumin through multivalent binding to glycogen for drug delivery in 2D and 3D multicellular constructs

“…It has been reported, that DOX increases the production of tumour necrosis factor (TNF), interleukin-1 (IL-1), interferon γ (IFNγ) and NO, therefore, increasing the tumoricidal potential of macrophage cells. 60,61 Another study 62 reported that the co-culture with Raw 264.7 cells reduced the viability and susceptibility to DOX of the three different cancer cell lines, which is consistent with our earlier findings.…”

Preparation of a PEGylated liposome that co-encapsulates l-arginine and doxorubicin to achieve a synergistic anticancer effect

“…21 NO production by macrophages incubated with Arg-LP NO production by macrophages using Arg as the substrate in response to lipopolysaccharides (LPS) stimulation has been reported. 18,22 Here, we evaluated NO production by macrophages incubated with Arg-LP. Macrophages were rst stimulated with LPS (1 mg mL À1 ) for 6 h to activate the expression of iNOS and then incubated with different amounts (dened as Arg concentration in mM) of Arg-LP for 48 h. The produced NO was detected by Griese reagent.…”

“…Following the successful preparation of Arg-LP, we sought to encapsulate Dox into Arg containing liposomes by using a remote loading method to yield an efficient drug delivery system with reduced side effects. Based on our previous studies, 18,23 we hypothesized that combining Arg and Dox in a single delivery system should afford an efficient synergistic anticancer effect. Remote loading of Dox into liposomes was performed by using a pH gradient between the internal and external solutions.…”

“…The experiments were conducted following the procedure of previous works 18,25 in polycarbonate cell culture inserts which consisted of two compartments separated by a porous membrane (0.4 mm pore size, catalog number 140620, Thermo Scientic Nunc). Colon cancer cells (CT-26) were cultured at the lower chamber at a density of 50 000 cells per well and RAW 264.7 were seeded into the upper chamber in 80 000 cells per well.…”

“…[14][15][16][17] We recently showed that supplementing Dox with Arg afforded a synergistic anticancer effect with the help of activated M1 macrophages in vitro. 18 Hence, in the present study, this synergistic effect was further exploited by co-encapsulating Arg and Dox in PEGylated liposomes (Dox-Arg-LP) to utilize the EPR effect. Arg was incorporated into the liposome using a hydration procedure and Dox was then efficiently encapsulated into the liposome through a remote loading strategy.…”