In Vitro and In Vivo Efficacy of a Novel Glucose–Methotrexate Conjugate in Targeted Cancer Treatment

Abstract

Methotrexate (MTX) is a commonly used antimetabolite, which inhibits folate and DNA synthesis to be effective in the treatment of various malignancies. However, MTX therapy is hindered by the lack of target tumor selectivity. We have designed, synthesized and evaluated a novel glucose–methotrexate conjugate (GLU–MTX) both in vitro and in vivo, in which a cleavable linkage allows intracellular MTX release after selective uptake through glucose transporter−1 (GLUT1). GLU–MTX inhibited the growth of colorectal (DLD-1), breast (MCF-7) and lung (A427) adenocarcinomas, squamous cell carcinoma (SCC-25), osteosarcoma (MG63) cell lines, but not in WI-38 healthy fibroblasts. In tumor cells, GLU–MTX uptake increased 17-fold compared to unconjugated MTX. 4,6-O-ethylidene-α-D-glucose (EDG), a GLUT1 inhibitor, significantly interfered with GLU–MTX induced growth inhibition, suggesting a glucose-mediated drug uptake. Glu-MTX also caused significant tumor growth delay in vivo in breast cancer-bearing mice. These results show that our GLUT-MTX conjugate can be selectively uptake by a range of tumor cells to cause their significant growth inhibition in vitro, which was also confirmed in a breast cancer model in vivo. GLUT1 inhibitor EDG interfered with these effects verifying the selective drug uptake. Accordingly, GLU–MTX offers a considerable tumor selectivity and may offer cancer growth inhibition at reduced toxicity.

The Effect of a New Glucose–Methotrexate Conjugate on Acute Lymphoblastic Leukemia and Non-Hodgkin’s Lymphoma Cell Lines

“…Moreover, the compound exhibited potent activity in low doses, comparable to free MTX. However, increased uptake of Glu-MTX apparently did not translate into a significantly higher cytotoxic and apoptotic effect, which serves as an indication that the conjugate is less cytotoxic, compared to free MTX, which in turn supports the results of our previous study [11]. Another possible explanation for this could be the fact that the drug uptake was measured after 6 h, while the apoptotic effect was evaluated after 48 h, which suggests that efflux of free MTX originated from Glu-MTX to the outside of the cells could have occurred.…”

“…Such a designed structure of the tested compound allows for the assumption that after penetrating into the cell, Glu–MTX undergoes degradation with the release of the active form of the drug (MTX) and metabolites such as carbon dioxide and the 1,2,3-triazole derivative of glucose 3 , and the latter one may degrade further over time ( Figure 1 ). An additional advantage of this conjugate is the fact that the glucose derivative 3 , containing the 1,2,3-triazole unit, released in the cell as a result of enzymatic hydrolysis, also has a slight cytotoxic activity [ 11 ]. Glycoconjugate Glu–MTX may be prepared in an efficient manner using one of the so-called click-chemistry reaction, the copper-catalyzed 1,3-dipolar cycloaddition of the azide to a terminal alkyne bond (CuAAC) in a variant developed by Sharpless and used for the synthesis of a wide range of biologically active compounds [ 12 , 13 , 14 , 15 ].…”

Folate-Targeted Monodisperse PEG-Based Conjugates Made by Chemo-Enzymatic Methods for Cancer Diagnosis and Treatment

“…Long-term cytotoxicity study showed ( Figure 2 c) that after treatment with FA 2 -dPEG-DOX 2 at 100 μM concentration (regarding DOX), viability values of the treated MDA-MB-231 cells were similar to the untreated control until 48 h. However, treatment with FA 2 -dPEG-DOX 2 reduced cell viability significantly after 48 h (compared to the control) and sustained at an approximately constant level for 168 h. The results show slow DOX release in the cell culture medium investigated. Similar studies often do not investigate simulated drug release profiles because they may not be predictive for in vivo conditions [ 15 , 16 , 17 , 18 , 19 , 20 ].…”

“…PDCs. Research carried out with various polymers, including PEG, showed promise due to increased water solubility and circulation time in the body, and multivalent attachment to receptors FR [ 15 , 16 , 17 , 18 , 19 , 20 ]. Despite the progress achieved in the field, several obstacles currently impede the clinical translation of PDC-based therapeutics [ 21 ].…”

Insulin-Like Growth Factor 1 (IGF-1) Signaling in Glucose Metabolism in Colorectal Cancer

“…These results confirmed the hypothesis that GLUT1 is active during the cellular uptake of GLU-MTX, with the absorption of the drug mediated by glucose. This conjugate was about 17-times more preferentially accumulated in SW-480 cells compared to free MTX [ 245 ].…”