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The AKT inhibitor MK-2206 is cytotoxic in hepatocarcinoma cells displaying hyperphosphorylated AKT-1 and synergizes with conventional chemotherapy.

Simioni C, Martelli AM, Cani A, Cetin-Atalay R, McCubrey JA, Capitani S, Neri LM - Oncotarget (2013)

Bottom Line: The inhibitor decreased cell viability and induced cell cycle arrest in the G0/G1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1.MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment.Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Advanced or recurrent HCC is frequently resistant to conventional chemotherapeutic agents and radiation. Therefore, targeted agents with tolerable toxicity are mandatory to improve HCC therapy and prognosis. In this neoplasia, the PI3K/Akt signaling network has been frequently shown to be aberrantly up-regulated. To evaluate whether Akt could represent a target for treatment of HCC, we studied the effects of the allosteric Akt inhibitor, MK-2206, on a panel of HCC cell lines characterized by different levels of Akt-1 activation. The inhibitor decreased cell viability and induced cell cycle arrest in the G0/G1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1. Moreover, MK-2206 induced apoptosis, as documented by Annexin V labeling, and also caused autophagy, as evidenced by increased levels of the autophagy marker LC3A/B. Autophagy was shown to be a protective mechanism against MK-2206 cytotoxicity. MK-2206 down-regulated, in a concentration-dependent manner, the phosphorylation levels of Akt-1 and its downstream targets, GSK3 α/β and FOXO3A. MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment. Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.

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Different levels of Akt-1 phosphorylation are detected in HCC cell lines and correlate with cell cycle block induced by MK-2206(A) Western blot analysis for Ser 473 p-Akt-1, total Akt-1 and PTEN in HCC cell lines. Fifty μg of protein was blotted to each lane. An antibody to β-actin documented equal lane loading. (B) Cell cycle in PLC and Mahlavu cells, treated with MK-2206, was analyzed by the Muse™ Cell Analyzer, according to the instrument protocol. The results are the mean ± s.d. of three different experiments. Asterisks indicate significant differences (p<0.05) in comparison with control (CTRL).
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Figure 1: Different levels of Akt-1 phosphorylation are detected in HCC cell lines and correlate with cell cycle block induced by MK-2206(A) Western blot analysis for Ser 473 p-Akt-1, total Akt-1 and PTEN in HCC cell lines. Fifty μg of protein was blotted to each lane. An antibody to β-actin documented equal lane loading. (B) Cell cycle in PLC and Mahlavu cells, treated with MK-2206, was analyzed by the Muse™ Cell Analyzer, according to the instrument protocol. The results are the mean ± s.d. of three different experiments. Asterisks indicate significant differences (p<0.05) in comparison with control (CTRL).

Mentions: We first analyzed the basal expression of Akt-1 and its phosphorylation status on Ser473 on a panel of human HCC cell lines (PLC, SNU387, Mahlavu, SNU449 and SNU475 cells). Akt-1 total amount was similar in the five cell lines examined (Fig. 1A). On the contrary, the phosphorylation status of the protein, as documented by Western blot analysis with an antibody to Ser 473 p-Akt-1, showed relevant differences: in PLC cells a negligible phosphorylation level of Akt-1 was observable, in SNU387 cells only a slight Akt-1 phosphorylation was detectable, whereas a significant Akt-1 phosphorylation was detectable in Mahlavu, SNU449 and SNU475 cells. SNU387 cells in comparison with PLC cells showed a decrease of PTEN protein. A lower expression in SNU449 or loss of PTEN protein in Mahlavu and SNU475 cell lines, respectively, was associated with Akt-1 hyperphosphorylation. Therefore, our initial observations suggested that Mahlavu, SNU449 and SNU475 cells displayed hyperactivated Akt-1 when compared with PLC and SNU387 HCC cell lines.


The AKT inhibitor MK-2206 is cytotoxic in hepatocarcinoma cells displaying hyperphosphorylated AKT-1 and synergizes with conventional chemotherapy.

Simioni C, Martelli AM, Cani A, Cetin-Atalay R, McCubrey JA, Capitani S, Neri LM - Oncotarget (2013)

Different levels of Akt-1 phosphorylation are detected in HCC cell lines and correlate with cell cycle block induced by MK-2206(A) Western blot analysis for Ser 473 p-Akt-1, total Akt-1 and PTEN in HCC cell lines. Fifty μg of protein was blotted to each lane. An antibody to β-actin documented equal lane loading. (B) Cell cycle in PLC and Mahlavu cells, treated with MK-2206, was analyzed by the Muse™ Cell Analyzer, according to the instrument protocol. The results are the mean ± s.d. of three different experiments. Asterisks indicate significant differences (p<0.05) in comparison with control (CTRL).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824526&req=5

Figure 1: Different levels of Akt-1 phosphorylation are detected in HCC cell lines and correlate with cell cycle block induced by MK-2206(A) Western blot analysis for Ser 473 p-Akt-1, total Akt-1 and PTEN in HCC cell lines. Fifty μg of protein was blotted to each lane. An antibody to β-actin documented equal lane loading. (B) Cell cycle in PLC and Mahlavu cells, treated with MK-2206, was analyzed by the Muse™ Cell Analyzer, according to the instrument protocol. The results are the mean ± s.d. of three different experiments. Asterisks indicate significant differences (p<0.05) in comparison with control (CTRL).
Mentions: We first analyzed the basal expression of Akt-1 and its phosphorylation status on Ser473 on a panel of human HCC cell lines (PLC, SNU387, Mahlavu, SNU449 and SNU475 cells). Akt-1 total amount was similar in the five cell lines examined (Fig. 1A). On the contrary, the phosphorylation status of the protein, as documented by Western blot analysis with an antibody to Ser 473 p-Akt-1, showed relevant differences: in PLC cells a negligible phosphorylation level of Akt-1 was observable, in SNU387 cells only a slight Akt-1 phosphorylation was detectable, whereas a significant Akt-1 phosphorylation was detectable in Mahlavu, SNU449 and SNU475 cells. SNU387 cells in comparison with PLC cells showed a decrease of PTEN protein. A lower expression in SNU449 or loss of PTEN protein in Mahlavu and SNU475 cell lines, respectively, was associated with Akt-1 hyperphosphorylation. Therefore, our initial observations suggested that Mahlavu, SNU449 and SNU475 cells displayed hyperactivated Akt-1 when compared with PLC and SNU387 HCC cell lines.

Bottom Line: The inhibitor decreased cell viability and induced cell cycle arrest in the G0/G1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1.MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment.Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy.

ABSTRACT
Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Advanced or recurrent HCC is frequently resistant to conventional chemotherapeutic agents and radiation. Therefore, targeted agents with tolerable toxicity are mandatory to improve HCC therapy and prognosis. In this neoplasia, the PI3K/Akt signaling network has been frequently shown to be aberrantly up-regulated. To evaluate whether Akt could represent a target for treatment of HCC, we studied the effects of the allosteric Akt inhibitor, MK-2206, on a panel of HCC cell lines characterized by different levels of Akt-1 activation. The inhibitor decreased cell viability and induced cell cycle arrest in the G0/G1 phase of the cell cycle, with a higher efficacy in cells with hyperphosphorylated Akt-1. Moreover, MK-2206 induced apoptosis, as documented by Annexin V labeling, and also caused autophagy, as evidenced by increased levels of the autophagy marker LC3A/B. Autophagy was shown to be a protective mechanism against MK-2206 cytotoxicity. MK-2206 down-regulated, in a concentration-dependent manner, the phosphorylation levels of Akt-1 and its downstream targets, GSK3 α/β and FOXO3A. MK-2206 synergized with doxorubicin, a chemotherapeutic drug widely used for HCC treatment. Our findings suggest that the use of Akt inhibitors, either alone or in combination with doxorubicin, may be considered as an attractive therapeutic regimen for the treatment of HCC.

Show MeSH
Related in: MedlinePlus