Antitumor activity of a novel antisense oligonucleotide against Akt1

Abstract

The AKT pathway is an important therapeutic target for cancer drug discovery as it functions as a main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers. In the present study, we found that an Akt1 antisense oligonucleotide (Akt1 AO) significantly downregulated the expression of AKT1 at both the mRNA and protein levels and inhibited cellular growth at nanomolar concentrations in various types of human cancer cells. Combined treatment of Akt1 AO with several cytotoxic drugs resulted in an additive growth inhibition of Caki-1 cells. The in vivo effectiveness of Akt1 AO was determined using two different xenograft nude mouse models. Akt1 AO (30 mg/kg, i.v. every 48 h) significantly inhibited the tumor growth of nude mouse subcutaneously implanted with U251 human glioblastoma cells after 27 days treatment. Akt1 AO (30 mg/kg, i.p continuously via osmotic pump) also significantly inhibited the tumor formation in nude mice implanted with luciferase-expressing MIA human pancreatic cancer cells (MIA-Luc) after 14 days of treatment. The luciferase signals from MIA-Luc cells were reduced or completely abolished after 2 weeks of treatment and the implanted tumors were barely detectable. Our findings suggest that Akt1 AO alone or in combination with other clinically approved anticancer agents should be further explored and progressed into clinical studies as a potential novel therapeutic agent.

(c) 2009 Wiley-Liss, Inc.

Figures

Fig. 1

Effect of Aktl AO on Aktl mRNA expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using Lipofectamine Plus for 3 h and incubated for 6 h with fresh media containing 10% FBS. Total RNA was isolated and Akt1 mRNA expression was determined using RT-PCR. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments and each RT-PCR reaction was performed in duplicate.

Fig. 2

Effect of Akt1 AO on AKT1 protein expression in various human cancer cells. The cells were transfected without (−) or with 0.3 µM of Akt1 AO (+) using Lipofectamine Plus for 3 h and incubated for 24 h with fresh media containing 10% FBS. Cellular protein was isolated and AKT1 protein expression was determined by Western blot analysis. β-Actin was determined as a loading control. Representative data are shown from two independent cell culture experiments.

Fig. 3

The sequence of Aktl AO is specific to inhibit Aktl mRNA expression in UMRC2 cells. The cells were transfected with 0.3 µM of Akt1 AO, sense sequence oligonucleotide of Akt1 AO, and mismatch sequence oligonucleotide using Lipofectamine Plus for 3 h and incubated for 6 h with fresh media containing 10% FBS. Total mRNA were isolated and Akt1 mRNA expression was determined using RT-PCR. β-Actin was determined as a loading control. Each RT-PCR reaction was performed in duplicate. Representative data are shown from two independent cell culture experiments.

Fig. 4

Combination effect of Akt1 AO with different cytotoxic drugs on the growth of Caki-1 cancer cells. The drugs were used at the following doses: a,b, 2 and 3 nM paclitaxel; c,d, 5 and 10 nM doxorubicin; e,f, 2 and 5 µM fluorouracil; g,h, 0.5 and 1 nM docetaxel, i,j 0.5 and 1.0 µM cisplatin. Data are expressed as percentage growth inhibition in reference to the growth of untreated control cells. Lipofectamine alone (no Akt1 AO) and 0.25% DMSO in media were used as a control for drugs except cisplatin. Lipofectamine alone and water were used as a control for cisplatin. The open portion of the bars represents the percentage growth inhibition values for Akt1 AO. The striped or squared portion of the bars represents the percentage growth inhibition values for the cytotoxic drugs as indicated in the respective legends. The height of the bars on the left represents the sum of the individual agents’ effects and the expected percentage growth inhibition if drugs are additive when used in combination. The total height of the solid bar indicates the actual observed growth inhibition when drugs were used in combination. The data represent means and standard errors of triplicate determination of at least two experiments.

Fig. 5

Effect of Akt1 AO on the human glioblastoma U251 tumor xenograft. Nude micebearing U251 tumorsweretreated bytail veil injections every other day for 3 weeks with either saline as vehicle control (○) or Akt1 AO at dose of 30 mg/kg (●) per injection. After Akt1 AO treatment for 3 weeks, the mice were observed for up to 30 more days to detect possible tumor regrowth. Data shown are average tumor weight ±SEM (n = 6–8 per group).

Fig. 6

Representative in vivo bioluminescence images of Aktl AO treatment on luciferase-expressing MIA (MIA-Luc) human pancreatic cancer cells. Nude mice were subcutaneously implanted with MIA-Luc cells at 2.5 × 106 in each flank area. Akt1 AO (30 mg/kg) was continuously and intraperitoneally infused using osmotic pump to the mice. The images on the left hand are the control and the images on the right hand are Akt1 AO treated. A: Control group: The images on the left is 1 week after the injection of MIA-Luc cells and the image on the right is 2 weeks after the injection of cells. B: Akt1 AO treatment group (30 mg/kg): The images on the left is 1 week after treatment with Akt1 AO and the image on the right is 2 weeks after treatment with Akt1 AO. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]