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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 1  |  Page : 208-212

Effects of the Akt inhibitor Src-homology 5 on proliferation and apoptosis of the laryngeal squamous cell carcinoma


1 Department of Otolaryngology, The Second Hospital of Shandong University, Jinan 250000, Shandong Province, China
2 Department of Health, The Second Hospital of Shandong University, Jinan 250000, Shandong Province, China

Date of Web Publication8-Mar-2018

Correspondence Address:
Dr. Tao Jia
Department of Otolaryngology, The Second Hospital of Shandong University, No. 247 Beiyuan Avenue, Jinan 250000, Shandong Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.199454

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 > Abstract 

Objective: The aim of this study was to investigate the effect of the Akt inhibitor Src-homology 5 (SH-5) on the proliferation and apoptosis of laryngeal squamous cell carcinoma cells (LSCC; Hep-2 cells) and to elucidate the possible mechanisms of such effects.
Materials and Methods: Hep-2 cells were treated with different concentrations of the Akt inhibitor SH-5. The inhibitory effect of SH-5 on cell proliferation was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, whereas apoptosis was detected by flow cytometric based on Annexin V/propidium iodide (PI) staining. In addition, the expression level of Akt protein was evaluated by Western blot analysis.
Results: MTT assay results revealed that SH-5 inhibited the proliferation of Hep-2 cells, with its greatest effect being observed at 2 μM. Apoptosis of Hep-2 cells increased following treatment with SH-5. Treatment of Hep-2 cells with SH-5 decreased the expression of Akt, and this effect was statistically significantly when compared with that in controls (P < 0.05).
Conclusion: SH-5 inhibited proliferation and induced apoptosis in the LSCC cell line Hep-2. These effects may be caused by inhibition of the phosphoinositide 3-kinase-Akt signaling pathway. We believe that our data will provide useful insights into LSCC target treatment and future research.

Keywords: Akt inhibitor Src-homology 5, apoptosis, Hep-2 cell line, laryngeal squamous cell carcinoma, proliferation


How to cite this article:
Jia T, Cao QQ, Chen XM, Xu FL. Effects of the Akt inhibitor Src-homology 5 on proliferation and apoptosis of the laryngeal squamous cell carcinoma. J Can Res Ther 2018;14:208-12

How to cite this URL:
Jia T, Cao QQ, Chen XM, Xu FL. Effects of the Akt inhibitor Src-homology 5 on proliferation and apoptosis of the laryngeal squamous cell carcinoma. J Can Res Ther [serial online] 2018 [cited 2019 Nov 17];14:208-12. Available from: http://www.cancerjournal.net/text.asp?2018/14/1/208/199454


 > Introduction Top


Squamous cell carcinoma (SCC) of the head and neck, one of the most frequent malignancies in the world, accounts for nearly 90% of all head and neck tumors,[1] of which a quarter are laryngeal SCCs (LSCCs).[2] LSCC, which comprises almost 2% of all malignancies worldwide, is an aggressive neoplasm of laryngeal epithelial origin.[3] In the absence of treatment, patients with LSCC have overall 1- and 2-year survival rates of only of 56.4% and 26.5%, respectively.[4] Currently, several cutting-edge treatment strategies have been proposed for LSCC such as laryngectomy, radiotherapy, or chemotherapy.[5] However, no treatment achieves a satisfactory therapeutic outcome, and the mortality rate of LSCC is still high (5-year survival rate of treated patients is 64%).[6] Therefore, there is urgency to discover novel biomarkers to distinguish patients with poor prognosis or at high risk of early recurrence, to guide chemotherapy and radiotherapy.[7]

With a better understanding of the genes related to pathogenesis and the molecular mechanisms underlying LSCC, the development of therapeutic inhibitors designed to target tumor cell signal transduction pathway has now become another possible effective approach to the treatment of LSCC. The phosphoinositide 3-kinase (PI3K)/Akt (also known as protein kinase B, PKB) signaling pathway is frequently disrupted in many human cancers, and therefore, may play a significant role in the occurrence and progress of LSCC. The PI3K/Akt signaling pathway regulates a variety of cellular processes including cell proliferation, survival, growth, and motility, which are critical for tumorigenesis.[8] Its hyperactivation is, therefore, often genetically selected for during tumorigenesis, and the normal cellular functions regulated by this pathway are adopted to promote the proliferation and survival of the cancer cells.[9] Specifically, Akt promotes cell survival by inhibiting pro-apoptotic proteins such as Bad, forkhead, and p53 and activating pro-survival proteins such as NF-κB. It also stimulates protein synthesis and cell growth by activating the mTOR pathway through inhibition of the tuberin/hamartin complex and promotes cell cycle progression by indirectly stabilizing cyclin D1 and Myc.[10],[11] Hence, inhibition of Akt could be a powerful means to reduce the activation of PI3K/Akt signaling pathway in tumors. Src-homology 5 (SH-5) is an inhibitor of Akt activation that functions without affecting the activation of kinases upstream of Akt or other kinases downstream of Ras.[12] As such, it induces apoptosis and kills a variety of cancer cell lines that contain high levels of active Akt and which are dependent on Akt for survival.[13] However, the effect of SH-5 in LSCC cells has not been determined.

Therefore, in this study, we examined the effect of the Akt inhibitor SH-5 on proliferation and apoptosis of the LSCC cell line Hep-2. We aimed to highlight the contribution of the Akt inhibitor SH-5 on the development of LSCC and provide experimental evidence leading to clinical application.


 > Materials and Methods Top


Cell line and culture conditions

Hep-2 cell lines derived from human LSCC and normal laryngeal squamous cells were kindly provided by the Laboratory of Head and Neck Cancer, Institute of Otolaryngology, Provincial Hospital Affiliated to Shandong University. Cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM)/F-12 containing 10% fetal bovine serum (FBS) (Gibco; Life Technologies, Carlsbad, CA, USA) and antibiotics (100 U/ml penicillin G, 100 μg/mL streptomycin and 250 ng/mL fungizone), which were purchased from Roth (Karlsruhe, Germany), at 37°C in a humidified incubator with 5% CO2 atmosphere (Shanghai Samsung Experimental Instrument Co. Ltd., Shanghai, China). When the cultures reached confluence (5 days), cells were treated with 0.05% trypsin/1 mM EDTA for 5 min at 37°C. Subsequently, the cell suspension was diluted with DMEM/F-12 supplemented with 10% FBS to give a concentration of 2 × 105 cells/mL, and plated in 12-well culture plates (1 mL/well). Culture medium was changed after 24 h and thereafter for every 3 days. Before performing analyses, cells were cultivated in the presence of differing concentrations of SH-5 for 4 h.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is widely performed to study the chemosensitivity or toxicity of drugs in human tumor cell lines.[14] The assay is based on the formation of a dark-colored formazan dye, which is formed by reduction of the tetrazolium salt (MTT) in metabolically active cells. The water-insoluble formazan dye forms crystals that can be dissolved in an organic solvent, and the amount of dye is determined semi-automatically in a microplate reader.[15] In this work, cells were cultivated in DMEM supplemented with 50 μg of MTT reagent/well (Roth, Karlsruhe, Germany) and incubated for 30 min at 37°C. Thereafter, the MTT solution was removed. After addition of 200 μL of SH-5 solution (prepared by dissolving solid SH-5 in dimethyl sulfoxide (DMSO) and then diluting this in phosphate-buffered saline (PBS) to give solutions of 0.5, 1, 2, 3, and 5 μM), the plates were incubated for 15 min at 37°C to dissolve the formazan crystals. Absorbance readings of DMSO extracts were performed at 560 nm, with a reference of 690 nm, using a Tecan Infinite F200 microplate reader (Crailsheim, Germany). Absorbance readings are related to the number of cells,[16] and the inhibition ratio of cell proliferation (I) was calculated as following:



Where, AT is the average absorbance of Hep-2 cells treated with SH-5, and AN is the average absorbance of untreated Hep-2 cells.

Annexin V/propidium iodide staining

Hep-2 cells, which were treated with SH-5 at 1 and 2 μM for 24 h, were washed three times with cold 10 mM PBS and resuspended in 1 × binding buffer (BD Biosciences, San Jose, CA, USA) at a concentration of 1 × 106 cells/mL. Cells were stained for Annexin V/APC and propidium iodide (PI), using an Annexin V apoptosis detection kit (Merck KGaA, Darmstadt, Germany). After mixing 100 μL of cell suspension with 5 μL Annexin V and PI, in the mixture was placed in the dark for 15 min, 500 μL of binding buffer was added, and the cells were incubated for a further 30 min at 4°C. Apoptotic cells were then detected using flow cytometric (Beckman Coulter, Inc., USA). In this regard, flow cytometric provides a way to measure both physical and chemical attribute of individual cells rapidly and with high accuracy and is widely used in the analysis of cell apoptosis.[17] Untreated Hep-2 cells were used as negative controls, and all experiments were repeated at least three times.

Western blot analysis

Western blot analysis was carried out to assess the expression level of Akt in Hep-2 cells with or without treatment with 2 μM SH-5. Following treatment, cultivated Hep-2 cells were lysed in ice-cold lysis buffer, which comprised 20 mM Tris-HCl (pH = 7.5), 10 μg/mL leupeptin, 20 μg/mL aprotinin, 1 mM phenylmethylsulfonyl fluoride, 5 mM EDTA, for 15 min. Cell lysates were clarified by centrifugation at 4°C for 10 min at 10,000 rpm, using a Biofuge PrimoR (Kendro, USA), to remove cell debris. The supernatant (cell lysate) was retrieved, and its protein content was measured by a Bio-Rad protein assay (Bio-Rad, Munich, Germany) with bull serum albumin as the standard. Cell lysate (containing 30 μg of protein) was mixed with sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min at 100°C. SDS-polyacrylamide gel electrophoresis was then performed to separate proteins, using a 10% bis-acrylamide/acrylamide gel for the separation.[18] Following transfer to a nitrocellulose membrane, nonspecific protein binding sites were blocked for 30 min in 5% fat-free dry milk-tris buffered saline (50 mM Tris-HCl, 250 mM NaCl, and 0.05% Tween 20; TBS). Membranes were further incubated for 12 h at 4°C with specific monoclonal antibodies for Akt (dilution 1:200) (Sigma, Germany) or β-actin (dilution 1:1,000) (Sigma, Germany). Blots were washed three times with TBS buffer and then incubated with a horseradish peroxidase-linked, anti-mouse, secondary antibody (dilution 1:5,000) in TBS. Immunopositive bands were revealed following incubation in chemiluminescent substrates along with exposure to film for a total of 2 h (Hyperfilm ECL Amersham Italia Srl, Milano, Italy). Protein bands were quantified by laser densitometer (Amersham Italia Srl, Milano, Italy).

Statistical analysis

Each experiment was carried out at least in triplicate, and the results were analyzed by Statistical Product and Service Solutions (SPSS Company, Chicago, IL, USA).[19] Data were expressed as mean ± standard deviation. Differences between groups were assessed by an unpaired, two-tailed Student's t-test.[20] P < 0.05 was considered a statistically significant difference.


 > Results Top


Src-homology 5 inhibits proliferation of Hep-2 cells

To measure cell growth, the MTT assay was performed for every 12 h on normal laryngeal squamous cells (Blank group) and Hep-2 cells were cultivated in the absence (control group) or presence of SH-5 (treated group), for a total of 72 h. MTT readings were proportional to the number of tumor cells in vitro, at least in the phase of exponential growth, as shown in [Figure 1]a. As time progressed, there was no increase in MTT signal for the Blank cells, whereas, for Control cells and the cells treated with SH-5, the absorbance was noted to increase sharply after 36 h, particularly for the tumor cells treated with SH-5 (1 and 2 μM). Moreover, because the concentration of SH-5 might significantly affect the efficiency of inhibition, we, therefore, examined the effect of different concentrations of SH-5 on the inhibition ratio and the correlation is shown in [Figure 1]b. As the concentration of SH-5 increased from 0 to 2 μM, the inhibition ratio was almost linearly dependent on concentration; thereafter, it remained constant. We conclude that 2 μM is the optimal SH-5 concentration required to inhibit proliferation of the LSCC cell line Hep-2.
Figure 1: Src-homology 5 inhibited cell proliferation of the laryngeal squamous cell carcinoma cell lines Hep-2 as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. (a) Hep-2 cells were cultivated for in the absence (control) or presence of Src-homology 5 (1 or 2 μM). Blank refers to normal laryngeal squamous cells. (b) Cell proliferation inhibition ratio (I) calculated at different concentrations of Src-homology 5

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Src-homology 5 induced apoptosis of Hep-2 cells

To investigate the effect of SH-5 on cell apoptosis, we performed Annexin V/PI staining and flow cytometric. The results showed that the percentage of apoptotic cells was significantly increased in Hep-2 cells treated with SH-5 than in untreated cells where 5.15% of the cells were apoptotic [Figure 2]. SH-5 at 1 μM increased the percentage of apoptotic to 17.91% and this was further increased to 31.71% by 2 μM SH-5. The increase in the percentage of apoptotic cells after SH-5 treatment was statistically significant difference (P< 0.05) when compared with untreated cells. Hence, SH-5 induces apoptosis in the LSCC Hep-2 cells.
Figure 2: Src-homology 5 induces apoptosis in Hep-2 cells. Representative flow cytometric dot plots for Hep-2 cells cultivated (a) in the absence or presence of Src-homology 5 (b) 1 μM Src-homology 5 and 2 μM (c)

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Src-homology 5 inhibited the activity of Akt

To evaluate the effect of SH-5 on the expression level of Akt in Hep-2 cells, a Western blot assay was implemented, and the results were shown in [Figure 3]. Akt was found to be overexpressed in Hep-2 cells when compared with the blank group, but its relative expression level was reduced following treating with SH-5. Specifically, we observed that the relative expression level of Akt in the control cells (8.56 ± 0.39) was significantly different with P < 0.05 compared with that in the blank group (3.29 ± 0.48). When the Hep-2 cells were treated with SH-5, we found that the expression level of Akt (5.34 ± 0.43) was decreased compared to untreated Hep-2 cells. We conclude that SH-5 reduces Akt expression in the LSCC cell line Hep-2.
Figure 3: Src-homology 5 treatment suppressed Akt expression in Hep-2 cells. (a) Western blot for Akt and β-actin in blank cells, control Hep-2 cells, and Src-homology 5 treated cells (b) quantitation of Akt expression (b)

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 > Discussions Top


Akt encodes a serine-threonine protein kinase, which is maintained in a catalytically inactive form in serum-starved primary, as well as immortalized fibroblasts. It can be activated by factors such as platelet-derived growth factor.[10] The activation is rapid and specific and occurs through the PI3K pathway.[21] Moreover, full activation of Akt leads to additional substrate-speciπc phosphorylation events in both the cytoplasm and nucleus,[22] and the kinase mediates numerous cellular functions including angiogenesis, metabolism, growth, proliferation, survival, protein synthesis, transcription, and apoptosis.[23] In addition, with the discovery of the important role of PI3K and Akt activation in tumorigenesis (e.g. breast cancer, gastric cancer, and LSCC), intense research has led to the discovery of cellular negative regulators of this pathway, including phosphatase and tensin homolog, the PH-domain leucine-rich repeat containing protein phosphatases 1/2, as well as small molecule regulators such as SH-5.[24],[25]

SH-5 is a specific inhibitor of Akt activation, without effects on other kinases. It is a structurally modified phosphatidylinositol ether lipid analog that binds to the PH domain of Akt [26] and potentiates the apoptosis induced by tumor necrosis factor.[27] Gills et al. reported that SH-5 inhibited Akt activation in nonsmall cell lung cancer H157 cells.[28] In addition, it has been shown to decrease levels of phospho-Akt in U87MG glioblastoma cells.[29] In our work, by Western blot analysis, we demonstrate that SH-5 inhibited the expression of Akt and that there was a statistically significant difference between Hep-2 cells treated with SH-5 and untreated cells (P< 0.05). This result is consistent with those in previous literature and confirmed the feasibility of our other experiments.

We further investigated the effect of SH-5 on proliferation and apoptosis of the LSCC cell line Hep-2. By MTT assay, we found that SH-5 prevents the proliferation of Hep-2 cells, however, although the inhibition was dose dependent up to 2 μM inhibitor, the effect plateaued above that concentration. In parallel, SH-5 also induced apoptosis in Hep-2 cells.


 > Conclusion Top


SH-5 inhibits the proliferation and induces apoptosis in the LSCC cell line Hep-2. These effects are most likely related to the inhibition of the PI3K-Akt signaling pathway, and we hope that this data will provide further insights into LSCC targeted treatment and future research.

Acknowledgments

This work was supported by Science and Technology Development Planning of Shandong Province (No. 2014GGE27476).

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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