Enhancing the Efficacy of Ara‑C through Conjugation with PAMAM Dendrimer and Linear PEG: A Comparative Study

1β-d-Arabinofuranosylcytosine (Cytarabine, Ara-C) is a key drug in the treatment of acute myeloid leukemia. Ara-C has a number of limitations such as a rapid deactivation by cytidine deaminase leading to the formation of a biologically inactive metabolite, Ara-U (1β-d-arabinofuranosyluracil), a low lipophilicity, and fast clearance from the body. To address these problems, we developed a conjugate in which hydroxyl-terminated PAMAM dendrimer, G4-OH [“D”] and PEG were used as carriers for the drug (Ara-C). The conjugates were synthesized using an efficient multistep protection/deprotection method resulting in the formation of a covalent bond between the primary hydroxyl group of Ara-C and dendrimer/PEG. The structure, physicochemical properties, and drug release kinetics were characterized extensively. ¹H NMR and MALDI-TOF mass spectrometry suggested covalent attachment of 10 Ara-C molecules to the dendrimer. The release profile of Ara-C in human plasma and in PBS buffer (pH 7.4) showed that the conjugates released the drug over 14 days in PBS, with the release sped up in plasma. In PBS, while most of the drug is released from PEG-Ara-C, the dendrimer continues to release the drug in a sustained fashion. The results also suggested that the formation of the inactive form of Ara-C (Ara-U) was delayed upon conjugation of Ara-C to the polymers. The inhibition of cancer growth by the dendrimer-Ara-C and PEG-Ara-C conjugates was evaluated in A549 human adenocarcinoma epithelial cells. Both dendrimer- and PEG-Ara-C conjugates were 4-fold more effective in inhibition of A549 cells compared to free Ara-C after 72 h of treatment

TMZ-Se triggers a greater autophagic response than TMZ, and inhibition of autophagy decreases the efficacy of TMZ-Se against glioma cells.

<p>(<b>A</b>) <i>Left panels</i>: LN229 and T98G cells were treated with TMZ or TMZ-Se for 48 h, and the level of LC3 was measured by Western blot analysis. Tubulin was used as a loading control. <i>Right panels</i>: LN229 and T98G cells were treated with TMZ-Se for 48 h in the presence or absence of bafilomycinA1, and the level of LC3 was measured by Western blot analysis. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were transfected with a GFP-LC3 plasmid, followed by treatment with TMZ-Se for 48h. At the end of treatment, the cells were observed under fluorescence microscope. (<b>C</b>) T98G cells treated with TMZ-Se or vehicle were harvested by trypsinization, fixed and embedded in spur resin. Ninety nm thin sections were cut and examined at 80 Kv with a JEOL 1200EX transmission electron microscope. (<b>D</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the absence or presence of 3-MA or bafilomycinA1, and cell viability was measured by MTT assay. (<b>E</b>) LN229 and T98G cells were transfected with an Atg5-targeted siRNA, and then treated with TMZ-Se for 48h. Cell viability was measured by MTT assay. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

Effects of TMZ-Se and TMZ on tumor growth in mouse glioma and melanoma xenograft models.

<p>(<b>A and B</b>) The human glioma cells LN229 (1×10⁵ cells in 15 µl of DMEM medium) were injected into the brains of 6-week-old male BALB/c nude mice at 4 mm depth under anesthesia with chloralic hydras (4%, 2ml/kg, ip). Three days after tumor cell implantation, mice were randomly divided into three groups (15 mice/group). Treatments were begun on day 4. TMZ-SE (15 mg/kg), TMZ (15 mg/kg) or vehicle (10% DMSO in saline) was given p.o. daily for 2 weeks. (<b>A</b>) At day 7 and day 21 after tumor cell implantation, the mice were euthanized, and the brains were fixed in 10% buffered formalin, embedded in paraffin, and then stained with hematoxillin-eosin (H&E). The images shown are the representative of 5 mice from each group; (<b>B</b>) The kaplan-Meier survival curves, n = 10; (<b>C</b>) Nude mice (swiss^nu/nu) were inoculated s.c. with UACC903 human melanoma cells (1×10⁶ cells/100 µl/mouse). When the tumors reached 50∼100 mm³ in volume, TMZ or TMZ-Se (15 mg/kg) was administered i.p. on days 1, 3, 5, 7 and 9. Tumor sizes and body weight of the animals were measured every other day. The differences between treatments were analyzed using a two-sample <i>t</i>-test. The survival curves of the tumor - bearing mice subjected to different treatments were estimated using Kaplan-Meier method and compared by log-rank statistic analysis.</p

TMZ-Se is more apoptogenic than TMZ in tumor cells.

<p>(<b>A</b>) LN229 and T98G cells were treated with 100 µM or 200 µM of TMZ or TMZ-Se for 48 h, and apoptosis was examined by flow cytometric analysis of Annexin V and 7-AAD staining. (<b>B</b>) LN229 and T98G cells were treated with TMZ or TMZ-Se for 48h, and the levels of caspse-9, caspase-3, PARP and survivin were measured by Western blot analysis. Tubulin was used as a loading control. (<b>C</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the absence or presence of Z-VAD, and cell viability was measured by MTT assay. (<b>D</b>) 1205LU and UACC cells were treated with TMZ or TMZ-Se for 48h, and the levels of PARP and survivin were measured by Western blot. Tubulin was used as a loading control. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

Chemical structures of TMZ-Se and TMZ.

<p>Chemical structures of TMZ-Se and TMZ.</p

TMZ-Se induces calpain-mediated degradation of Beclin 1.

<p>(<b>A</b>) LN229 and T98G cells were transfected with Beclin1siRNA followed by TMZ-Se treatment, and the levels of LC3, Beclin 1 and PARP were examined by Western blot. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were transfected with a Beclin 1-targeted siRNA, followed by treatment with TMZ-Se for 48h. Cell viability was measured by MTT assay. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

TMZ-Se induces calpain-mediated degradation of Beclin 1.

<p>LN229 and T98G cells were treated with 100 or 200 µM of TMZ or TMZ-Se for 48h, (<b>A</b>) the levels of Beclin 1 and Bcl-2 were measured by Western blot. Tubulin was used as a loading control; (<b>B</b>) the expression of <i>beclin1</i> mRNA was measured by qRT-PCR. (<b>C</b>) LN229 and T98G cells were treated with 100 or 200 µM of TMZ-Se for 48h in the presence or absence of 10µM of MG132, and the level of Beclin 1 were measured by Western blot. Tubulin was used as a loading control. (<b>D</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the presence or absence of 20µM ALLM (<i>left panel</i>) or 10µg/ml E64D (<i>right panel</i>), and the level of Beclin1 was measured by Western blot. Tubulin was used as a loading control. (<b>E</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the presence or absence of 20µM ALLM or 10µg/ml E64D, and cell viability was measured by MTT assay. **<i>p</i> < 0.01.</p

Effects of TMZ-Se and TMZ on phosphorylation of H2AX, and on activity of Akt-mTOR-S6K pathway.

<p>(<b>A</b>) T98G, LN229, 1205LU and UACC903 cells were treated with 100 µM or 200 µM of TMZ or TMZ-Se for 48h, and the level of phospho-H2AX was measured by Western blot. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were treated with TMZ or TMZ-Se for 48 h, and the levels of p-Akt, Akt, p-mTOR, mTOR, p-S6K, and S6K were measured by Western blot analysis. Tubulin was used as a loading control.</p

Additional file 1 of Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB

Supplementary Material 1