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. 2012 Apr 1;11(7):1374–1382. doi: 10.4161/cc.19798

Metformin suppresses growth of human head and neck squamous cell carcinoma via global inhibition of protein translation

Arron Sikka 1, Manjinder Kaur 1, Chapla Agarwal 1,2, Gagan Deep 1,2, Rajesh Agarwal 1,2,✉
PMCID: PMC3350878  PMID: 22421144

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth leading cancer in the world; the main risk factors are alcohol and tobacco use. Advancements in therapies have yet to improve the prognosis of HNSCC. the connection between diabetes and cancer is being recognized, and metformin has been shown to decrease cancer incidence in diabetic patients. Accordingly, here, for the first time, we investigated metformin's efficacy on the growth and viability of human HNSCC FaDU and Detroit 562 cells. our results show that metformin treatment (5–20 mM) dose-dependently inhibits the growth of both cell lines. In FaDU cells, metformin caused 18–57% and 35–81% growth inhibition after 48 and 72 h treatments, respectively. Similarly, in Detroit 562 cells, 48 and 72 h metformin treatment resulted in 20–57% and 33–82% inhibition, respectively. Mechanistically, metformin caused G1 arrest, which coincided with a decrease in the protein levels of Cdks (2, 4 and 6), cyclins (D1 and e) and Cdk inhibitors (p15, p16, p18 and p27) but no change in p19 and p21. Metformin also decreased the levels of oncogenic proteins Skp2 and β-Trcp. In other studies, metformin decreased the phosphorylation of 4E-BP1 at Ser65, Thr37/46 and Thr70 sites but drastically increased the phosphorylation of EF2 at Thr56 and AMPK at Thr172, which results in global translational inhibition. In summary, the observed wide spectrum of mechanistic effects of metformin on HNSCC cells provides support for the anticancer capability of the drug and its potential use in future therapies.

Key words: metformin, head and neck cancer, cell viability, growth inhibition, cell cycle arrest, chemoprevention

Introduction

Metformin (1,1-Dimethylbiguanide hydrochloride) is the most commonly prescribed oral antidiabetic drug for the treatment of diabetes mellitus type 2.1 Contrary to phenformin, this drug is rarely associated with lactic acidosis.1 In a report by Stang et al., there were only nine cases of lactic acidosis per 100,000 person-years in patients taking metformin.2 In addition, metformin is also prescribed for the treatment of polycystic ovarian syndrome (PCOS) and obesity.3,4 Several mechanisms have been proposed for the antidiabetic effects of metformin, such as suppression of hepatic gluconeogenesis, increase in peripheral glucose uptake and reduction in insulin resistance.5 Other mechanisms include upregulation of GLUT-4 mRNA expression, with resultant increase in GLUT-4 protein content,6 enabling greater glucose transport within cells. In addition, metformin inhibits the activity of NADH dehydrogenase (complex I) of the respiratory chain,6 leading to energy restriction and an increase in anaerobic respiration within the cell. In response to decreased energy production, there is an increase in glycolytic activity inside the cell accompanied by increased glucose uptake.

The metabolic stress imposed by metformin leads to a high AMP/ATP ratio, thereby triggering the activation of AMP-activated protein kinase (AMPK) by the upstream LKB1 protein kinase.7 Activation of AMPK has several effects, all revolving around the priority of the cell to preserve energy and create more ATP.8 AMPK activation phosphorylates the TSC2 tumor suppressor and raptor, an mTORC1 binding subunit, which results in inhibition of the mammalian target of rapamycin (mTOR) pathway.9 The mTOR pathway plays a key role in cell growth control and metabolism,9 which are important events in cancer progression. Therefore, metformin may be utilized for the metabolic management of cancer.

Cancers of the head and neck affect the oral cavity, salivary glands, paranasal sinuses and nasal cavity, pharynx, larynx and the lymph nodes in those regions.10 It is estimated that there are approximately 600,000 new cases of head and neck cancer worldwide, with 47,560 diagnosed in the United States.10,11 Head and neck cancer is highly fatal, and because it is diagnosed at advanced stages, the five year survival rate is at a dismal 35–50%.12 Risks of developing head and neck cancer are positively associated with tobacco and alcohol use, which account for nearly 75% of cases.11 Another important risk factor, independent of tobacco and alcohol use, is infection with human papilloma virus (HPV), where HPV may assist in the development of head and neck cancer.13 Histologically, squamous cell carcinoma accounts for over 85% of head and neck cancers, affecting primarily the oral cavity, larynx and pharynx.11 Because head and neck cancer is primarily a tobacco-linked cancer, it is estimated that the number of cases will decrease in North America and western Europe, due to the decreased tobacco use in those regions.11 However, the number of head and neck cancer cases in developing countries is expected to continue to increase,11 resulting in an overall increase in the number of cases worldwide.

Surgery and radiation therapy are the first lines of treatment for head and neck cancer, while chemotherapy is used for later stages.14 During surgery, non-cancerous tissue is often removed because of its close proximity to the tumor,15 and therefore radiation therapy results in a greater probability of organ preservation.14 However, resistance to radiation and recurrence after therapy are major drawbacks for patients with head and neck cancer and remains a point of emphasis for newer strategies of treatments in the future.16

Patients taking metformin have shown lower incidence of cancer in various retrospective epidemiological studies.17–19 Additionally, metformin has been shown to reduce tumor burden and average tumor volume in mice20 as well as increase lifespan and delay tumor onset.21–23 These anticancer effects, along with the activation of AMPK and inhibition of mTOR, might contribute to the chemopreventive potential of metformin. However, current literature lacks information detailing the mechanistic basis behind the lower incidence of cancer by metformin intake. In the present study, for the first time, we examined the anticancer effects and the associated mechanisms of metformin on head and neck squamous cell carcinoma (HNSCC) cell lines, FaDU and Detroit 562. Our results clearly suggest the promising anticancer effects of metformin against HNSCC through targeting the regulators of global translational machinery.

Results

Metformin inhibits the growth of human HNSCC cells.

Initially, we determined the effect of metformin on the growth and viability of both the head and neck cancer cell lines, FaDU and Detroit 562. After the 24 h treatment of FaDU cells, total cell number decreased by approximately 12% at the highest dose of metformin (20 mM), but the decrease in neither total nor live cell numbers was statistically significant (Fig. 1A and B). During the 48 and 72 h treatments, however, total and live cell numbers decreased significantly at all concentrations of metformin (p < 0.001), resulting in an 18–57% and 35–81% decrease in live cell number, respectively (Fig. 1A and B). The Detroit 562 cell line showed similar results but was a little more sensitive during the 24 h treatment, in which there was a significant decrease in total and live cell number when treated with 10 and 20 mM metformin (p < 0.05) (Fig. 1A and B). In the 48 h treatment, the decrease in total and live cells was slightly more significant with 5 mM metformin (p < 0.01), and had the greatest significance with 10 mM and 20 mM metformin (p < 0.001) (Fig. 1A and B). When Detroit 562 cells were treated for 72 h, all doses of metformin decreased total and live cells with the greatest significance (p < 0.001) (Fig. 1A and B). The 48 and 72 h treatments ultimately lead to a 20–57% and 33–82% decrease in live cells, respectively (Fig. 1A and B). The significant decreases in total number of cells clearly indicated the cell growth inhibitory effect of metformin in both HNSCC cell lines.

Figure 1.

Figure 1

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Metformin inhibits the growth of human HNSCC FaDU and Detroit 562 cells. Approximately 5,000 cells/cm2 were plated overnight on each 60 mm dish and then treated with metformin at concentrations of 0, 5, 10 or 20 mM for 24, 48 or 72 h. Total cell number (A), live cell number (B) and % dead cells (C) were analyzed as detailed in Methods. The data shown are mean values of three independent samples ± standard deviation. *(p < 0.05), #(p < 0.01), $(p < 0.001) vs. control.

The effect of metformin on causing cell death was also analyzed and presented as a percentage of dead cells to the total cell numbers in each case. In the FaDU cell line, the 24 h time point showed the greatest proportion of dead cells, i.e., 8% of total cells; none of the treatments at 48 and 72 h exceeded 6% death (Fig. 1C). In Detroit 562 cells, none of the treatments led to a statistically significant difference as compared with the control, thereby showing that metformin did not induce death in Detroit 562 cells (Fig. 1C). Together these results suggest that the growth inhibition caused by metformin treatment is not due to a reduction in cell viability in both FaDU and Detroit 562 cell lines.

Metformin induces cell cycle arrest in human HNSCC cells.

Based on strong growth inhibitory effects of metformin in two different human HNSCC cell lines without any noticeable cell death effects, we focused our efforts on deciphering the mechanism of cell growth inhibition by metformin. First, we analyzed the effect of metformin on the cell cycle progression of both HNSCC cell lines. As shown in Figure 2, metformin halted cell cycle progression, thereby providing insight to its growth-inhibitory effects. In the FaDU cell line, all treatments caused a dose- and time-dependent increase in G1-phase cell population (p < 0.001), which was mostly at the expense of the S-phase population (Fig. 2A and B).

Figure 2.

Figure 2

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Metformin blocks the cell cycle progression of human HNSCC FaDU and Detroit 562 cells in the G1-phase. After cells were plated overnight (105,000 cells/plate), they were treated with 0, 5, 10, 15 or 20 mM metformin for 24 (A) and 48 h (B). Thereafter, cell pellets were collected and incubated at 4°C for 24 h in saponin/propidium iodide solution and examined using flow cytometry as described in Methods. Values are means derived from three independent samples ± standard deviation. *(p < 0.05), #(p < 0.01), $(p < 0.001) vs. control.

Similarly, for Detroit 562 cells, 24 and 48 h metformin treatment led to a significant dose- and time-dependent increase in the G1-phase cell population (p < 0.001) (Fig. 2A and B). The 24 h treatment also caused a dose-dependent decrease in S phase and a marginal decrease in G2/M-phase cell population, while the 48 h treatment had a dose-dependent decrease in G2/M phase but did not change the S-phase population (Fig. 2A and B).

Metformin affects protein expression of cyclins, Cdks and CKDIs in human HNSCC cells.

Cdk/cyclin complexes interact in order to enable cells to progress past the G1 phase of the cell cycle.24 Meanwhile, accumulation of CDKIs of both Cip/Kip and INK4 family proteins inhibit that progression and prevent cells from going beyond a specific cell cycle phase depending on the signal driving the cell cycle progression.24 Since metformin caused G1-phase cell cycle arrest, we subsequently focused on assessing its effect on the cyclin-dependent kinase (Cdk), cyclin-dependent kinase inhibitor (CDKI), INK4 and cyclin protein levels associated with G1-phase progression.25 Metformin treatment for 24 and 48 h caused a strong and dose-dependent decrease in cyclin D1 and cyclin E levels in both FaDU and Detroit 562 cell lines (Fig. 3). For the FaDU cell line, Cdk2 did not appear to change during the 24 h treatment but slightly decreased during the 48 h treatment (Fig. 3). Meanwhile, Cdk2 levels showed a strong decrease during the 24 and 48 h treatment for Detroit 562 cells (Fig. 3). Cdk4 expression, however, decreased in a dose-dependent manner for both time points in both cell lines, with the effect being considerably stronger in the 48 h treatment (Fig. 3). Cdk6 levels did not change during the 24 h treatment of either cell line, but both showed an apparent decrease during the 48 h treatment (Fig. 3). INK4B/p15 levels only decreased with a dose of 20 mM metformin in Detroit 562 cells but significantly decreased with all metformin treatment in FaDU cells (Fig. 3). INK4A/p16 decreased significantly, and in a dose-dependent manner, in both the 24 and 48 h metformin treatments for FaDU cells (Fig. 3). In Detroit 562 cells, INK4A/p16 did not change during the 24 h treatment but showed a clear decrease after 48 h (Fig. 3). INK4C/p18 also decreased in both time points for FaDU cells (Fig. 3). In Detroit 562 cells, INK4C/p18 only showed a decreased with 20 mM metformin in the 24 h treatment but decreased in a dose-dependent manner in the 48 h treatment (Fig. 3). For both FaDU and Detroit 562 cells, INK4D/p19 did not appear to change with the treatment of metformin (Fig. 3). In FaDU cells, Cip1/p21 expression decreased with metformin treatment, although the effect was not strong (Fig. 3). In Detroit 562 cells, however, Cip1/p21 did not appear to change with metformin treatment (Fig. 3). For both cell lines, Kip1/p27 levels only showed a decrease with 20 mM treatment of metformin during the 24 h treatment (Fig. 3). The 48 h treatment, however, showed a clear and dose-dependent decrease of Kip1/p27 (Fig. 3). Overall, the decrease in expression of cyclin D1, cyclin E, Cdk2, Cdk4, Cdk6, INK4A/p16, INK4C/p18 and Kip1/p27 with metformin treatment was not HNSCC cell line-specific.

Figure 3.

Figure 3

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Metformin decreases the protein levels of cell cycle regulators in human HNSCC FaDU and Detroit 562 cells. FaDU and Detroit 562 cells were plated at approximately 60% confluency and then treated with 0, 5, 10 or 20 mM metformin for 24 and 48 h. Thereafter, cells were collected, lysates prepared, and western analysis was performed for cyclin D1, cyclin E, Cdk2, Cdk4, Cdk6, p15, p16, p18, p19, Cip1/p21 and Kip1/p27 levels as detailed in methods. β-actin was used as a loading control.

Metformin inhibits cellular protein translation machinery.

Protein levels of cyclins, Cdks and CDKIs can be regulated at transcriptional, translational or posttranslational levels. Since we observed a consistent decrease in the cyclin, Cdk and CDKI protein levels by metformin, we next assessed whether this involved a posttranslational mechanism such as proteosomal degradation. Therefore, we conducted a cycloheximide chase experiment in both FaDU and Detroit 562 cells in order to examine the effects of metformin on the half-life of the cell cycle regulatory proteins. Cells were treated with cycloheximide or a combination of cycloheximide and metformin to compare the effects to the control. Based on the density of the bands, the chase experiment showed no consistent change in the half-life of Cdk2, Cdk4, Cdk6, cyclin D1, cyclin E and Kip1/p27 with or without the addition of metformin (data not shown), suggesting that the observed decrease in the protein levels of these molecules is not due to an alteration in posttranslational mechanisms by metformin.

Two F-box proteins, β-transducin repeat-containing protein (β-Trcp) and Skp2, have gained much attention in regards to cancer progression. Both are known to cause unregulated degradation of their protein substrates and primarily contribute to the degradation of cell cycle proteins.26 In both cell lines, the expression of β-Trcp and Skp2 decreased during both 24 and 48 h treatments with metformin. β-Trcp expression appeared to not be affected by 5 mM metformin for either time point and decreased slightly at 10 mM metformin during the 24 h treatment for FaDU cells (Fig. 4). However, 10 mM metformin at 48 h for Detroit 562 and 20 mM metformin at both time points drastically decreased β-Trcp levels (Fig. 4). Skp2 decreased in a dose-dependent manner for both time points, although the effect was more significant during the 48 h treatment (Fig. 4). These results further corroborate the findings that metformin does not decrease protein levels via posttranslational mechanisms.

Figure 4.

Figure 4

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Metformin decreases the protein levels of oncogenic proteins β-trcp and SKp2 in human HNSCC FaDU and Detroit 562 cells. FaDU and Detroit 562 cells were plated at approximately 60% confluency, and then treated with 0, 5, 10 or 20 mM metformin for 24 and 48 h. thereafter, cells were collected, lysates prepared, and western analysis was performed for β-trcp and Skp2 levels as detailed in methods. β-actin was used as a loading control.

Because the effects of metformin were not found to be post-translational, we next focused on examining the mechanism(s) metformin could target to inhibit translation. First, we assessed the phosphorylation of 4E-BP1, a eukaryotic translation initiation factor with four phosphorylation sites that enable translational initiation.27 The expression of p4E-BP1 (Ser65) in both cell lines did not appear to change during the 24 h treatment but drastically decreased during the 48 h treatment in FaDU cells; a minimal effect was observed in Detroit 562 cells (Fig. 5). Metformin, however, strongly decreased the protein levels of p4E-BP1 (Thr37/46) in a dose-dependent manner in both cell lines (Fig. 5). Furthermore, there was a slight reduction in protein levels of p4E-BP1 (Thr70) during the 24 h treatment, while the 48 h treatment led to a greater decrease in both cell lines (Fig. 5). It is noteworthy that all the observed changes in the phosphorylation levels of 4E-BP1 at different sites were not due to a change in total 4E-BP1 protein levels (Fig. 5). The upstream negative regulator of 4E-BP1 is the energy-sensing protein AMPK; thus, to assess whether the constant decrease in all the protein levels is a result of the activation of AMPK, we next analyzed pAMPK (Thr172) levels. This was also done to assess if the activation of AMPK occurs upstream of 4E-BP1. We found that the phosphorylation of AMPK increased during all time points and treatments of metformin in both cell lines without any changes in total protein levels of AMPK, suggesting the role of AMPK activation in growth inhibition of HNSCC cells via inhibition of protein translation (Fig. 5).

Figure 5.

Figure 5

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Metformin targets the regulators of global translational machinery in human HNSCC FaDU and Detroit 562 cells. FaDU and Detroit 562 cells were plated at approximately 60% confluency and then treated with 0, 5, 10 or 20 mM metformin for 24 and 48 h. thereafter, cells were collected, lysates prepared, and western analysis was performed for p4E-BP1 (Ser65), p4E-BP1 (Thr37/46), p4E-BP1 (Thr70), 4E-BP1, pAMPK (Thr172), AMPK, pEF2 and EF2 levels as detailed in methods. β-actin was used as a loading control.

To assess whether metformin only inhibits the initiation step of translation, we also examined the protein levels of elongation factor (EF2) and its phosphorylation at the Thr56 site. EF2 activation is required to synthesize the proteins necessary for the G1-to S-phase transition, and because of its key contribution to cell cycle progression and cell growth, we focused on assessing the effect of metformin on its activity.28 In both cell lines, the inhibitory phosphorylation of EF2 (Thr56) significantly increased in a dose-dependent manner for the 24 and 48 h treatments; total EF2 protein levels only slightly decreased with such treatments (Fig. 5).

Discussion

The low survival rate associated with head and neck cancer is not only due to poor prognosis of the disease but also because of the anatomical/physiological constrains in this part of the body. This makes surgery an undesirable option, as it can result in changes in speech, inability to swallow, clotting disorders and damage to sinuses, glands and lymph nodes.29,30 Robot-assisted surgery is a new strategy that minimizes the side-effects of surgery, but it is still in the investigational stages.31 Nonetheless, there is still an increased need to find non-invasive strategies to limit the growth or prevent the occurrence of HNSCC. Metformin, a biguanide antidiabetic drug, has been found to lower the risk of cancer in diabetic patients. Multiple mechanisms are being proposed for metformin efficacy against cancer.23,32–37 To the best of our knowledge, we report here for the first time the anticancer efficacy of metformin and the associated mechanism of action in human HNSCC cells. We specifically selected FaDU and Detroit 562 HNSCC cells to determine metformin efficacy, as these cells encompass the molecular defects attributed to head and neck cancer. For example, SMAD4 deficiency and overexpression of EGFR and cyclin D1 are often observed in HNSCC and are associated with poor prognosis, and many EGFR based therapies have shown poor response in patients.38–41 So new targets are being tested which can improve the response of these therapies.42 SMAD4 is a tumor suppressor gene responsible for maintaining the anti-proliferative TGFβ signaling in cells,42 and the suppression of this pathway leads to tumor progression and metastasis.43

Aberrant SMAD4 signaling has been found to be associated with aggressive tumor pathology and recurrence of HNSCC.44 Both FaDU and Detroit 562 HNSCC cells harbor aberrant SMAD4 and overexpress EGFR and cyclin D1, and therefore our results in these cell lines are relevant to human disease conditions.

In our study, we observed that metformin causes strong growth inhibition of human HNSCC cells without causing any prominent cell death, and the growth inhibition was primarily due to G1-phase cell cycle arrest. The G1 phase of the cell cycle is controlled by a dynamic interaction between cyclins (D1 and E), Cdks (2, 4 and 6) and Cdk inhibitors (Cip/Kip and the INK4 family). During G1-phase transition, Kip1/p27 levels decrease in order to allow cyclin-Cdk complexes to begin transcribing genes needed for G1-S progression.24 Similarly, the expression of INK4 family members (p15, p16, p18 and p19) is important during early stages of the G1-phase.45 The increased expression of Cdk inhibitors, or their enhanced binding with cyclin/Cdk complexes, results in inhibition of Cdk kinase activity and accumulation of cells in G1-phase.46 However, our data shows that metformin causes a decrease in the expression of not only Cdks and cyclins, but also Cdk inhibitors in HNSCC cells. Importantly, the observed decrease in p27 levels in HNSCC cell lines caused by metformin was contrary to other published reports, where accumulation of p27 protein was observed upon treatment of cancer cells with metformin.47–49 Accordingly, we conducted additional studies to define the mechanisms involved in the observed decrease in cell cycle regulatory molecules by metformin, and found that the decrease in Cdks, cyclins and CDKIs by metformin occurred independently of the proteosomal pathway. The F-box protein, Skp2, is a key regulator of the cell cycle and is primarily involved in the degradation of CDKIs, specifically p27 along with other Cdks and cyclins.26,50,51 Skp2 is generally overexpressed in most cancers.51 In our study, metformin decreased the levels of Skp2 in conjunction with Cdks and p27. Like Skp2, β-Trcp plays a role in degrading cell cycle regulators,52 and an increase in expression of β-Trcp contributes to the proteolysis of cell cycle proteins.53 These proteins target tumor suppressors such as p27, PDCD4, IκB, p57, etc. for degradation and are thus oncogenic in nature.26 Again, we found that metformin decreases β-Trcp levels in both HNSCC cells. These observations suggest that the observed decrease in cell cycle protein levels by metformin is independent of both Skp2- and β-Trcp-mediated degradation. However, metformin-mediated decrease in these oncogenic proteins might contribute to the cancer preventive efficacy of metformin.26 Furthermore, the cycloheximide chase experiment did not show any change in the half-life of proteins (cyclins and Cdks) with metformin treatment, thereby underscoring the above findings that metformin does not enhance the proteasomal degradation of these proteins. In order for cells to advance from the G1- to S phase of the cell cycle, accumulation and activation of cyclins and Cdks is necessary to form the Cdk-cyclin complexes required for DNA replication.25,45 Since metformin significantly decreases the expression of cyclins and Cdks, cells arrest at the G1 phase in spite of decreased expression of CDKIs. Substantial decrease in cyclin D1 is noteworthy considering that HNSCC cells overexpress cyclin D1 protein.39 Similar findings were observed in prostate cancer cells, where it was reported that the decrease in cyclin D1 is responsible for metformin's anticancer effects.48

As mentioned earlier, metformin activates AMPK, which subsequently leads to the inhibition of mTOR;9 by that mechanism, metformin is an inhibitor of the mTOR pathway, which is a key contributor to the growth and advancement of cancer. 4E-BP1 is downstream of mTOR, and its activation is required for protein synthesis.54 The phosphorylation of 4E-BP1 allows the initiation factor eIF4E to begin cap-dependent translation.27 Metformin inhibited the phosphorylation at all four sites of 4E-BP1 (Thr37, Thr46, Thr70 and Ser65), which could adversely affect the eIF4E release and translation initiation. This finding is especially significant, because HNSCC cells overexpress eIF4E54 and thus have an enhanced protein synthesis rate. Elongation factor 2 (EF2) also plays a role in translation and translocates codons from the A site to the P site of the ribosome and is inactivated by phosphorylation at the Thr56 site by EF2 kinase.28 Metformin treatment also resulted in a dramatic increase in the phosphorylation of EF2 with a marginal decrease in total EF2 levels. Hence, metformin inhibits both the initiation and elongation steps and results in global translational inhibition in HNSCC cells. Thus, the observed decrease in the protein expression of Cdks, cyclins, CDKIs, Skp2 and Trcp by metformin could be due to its inhibitory effect on cellular translational machinery.

In conclusion, the results of the present study, for the first-time, show strong cell growth-inhibitory potential of metformin through targeting the regulators of global translational machinery in two different human HNSCC cell lines representing clinical HNSCC, supporting its potential usefulness in future therapies to control human HNSCC.

Materials and Methods

Reagents.

1,1-Dimethylbiguanide hydrochloride (metformin) (Sigma Aldrich, Catalog #D15,095-9) cycloheximide (Sigma Aldrich C7698) dimethyl sulfoxide (DMSO) (Sigma Aldrich 154938), β-actin antibody (Sigma Aldrich A-2228), p16 antibody (Cell Signaling Technology 4824), pACC antibody (Cell Signaling Technology 3661), ACC antibody (Cell Signaling Technology 3662), pAMPK (Thr172) antibody (Cell Signaling Technology 2535), AMPK antibody (Cell Signaling Technology 2532), p4EBP1 (Thr37/46) antibody (Cell Signaling Technology 2855), p4EBP1 (Ser65) antibody (Cell Signaling Technology 9451), p4EBP1 (Thr70) antibody (Cell Signaling Technology 9455), 4EBP1 antibody (Cell Signaling Technology 9452), pEF2 antibody (Cell Signaling Technology 2331) and EF2 antibody (Cell Signaling Technolog 2332). Cip1/p21 antibody (Millipore 05-345), p15 antibody (Millipore 05-430), cyclin D1 (Santa Cruz Biotechnology sc-718), cyclin E (Santa Cruz Biotechnology sc-198), Cdk2 (Santa Cruz Biotechnology sc-163), Cdk4 (Santa Cruz Biotechnology sc-749), Cdk6 (Santa Cruz Biotechnology sc-177), Skp2 (Santa Cruz Biotechnology sc-7164) and β-Trcp antibodies (Santa Cruz Biotechnology H-85), Anti-p18 antibody (Abcam ab85605), anti-p19 antibody (PharMingen 65911A) and p27 (NeoMarkers, Inc., MS-256-P).

Cell culture and treatments.

FaDU and Detroit 562 cell lines are human squamous cell carcinomas of the hypopharynx.55,56 Both FaDU and Detroit 562 were acquired from the American Type Culture Collection. Each cell line was cultured at 37.0°C and 5% CO2 in DMEM medium with 5% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. Both cell types were treated with metformin once they were plated for a minimum of 18 h and grown to ∼60% confluency. Metformin was dissolved directly into the medium at concentrations ranging from 5–20 mM for a period of 24–72 h.

Cell viability assay.

After determining the density of cells using a hemocytometer, approximately 5,000 cells/cm2 were plated on 60-mm plates. After 24, 48 and 72 h of treatments with metformin, both adherent and non-adherent cells from each plate were collected via brief trypsinization and centrifuged in order to get the cells in pellet form. Cell pellets were washed with ice-cold PBS. Cells were then suspended into an equal volume of ice-cold PBS and stained with trypan blue and counted with a hemocytometer using a light microscope. Unstained bright cells were counted as live, and cells that stained blue were considered dead.

Cell cycle analysis.

Both cell lines were either plated with plain medium (control) or treated with metformin (5–20 mM). After 24 and 48 h of treatment, cells were collected via brief trypsinization and centrifuged in order to pellet them down. After washing the pellet with 4°C PBS, the cell pellets were then combined with 500 µL of solution containing 25 µg/mL propidium iodide, 0.3% (w/v) saponin, 10 µg/mL RNase A and 0.1 mmol/L EDTA, then stored in dark at 4°C for 24 h. The samples were then analyzed by Flow Cytometry Core at the University of Colorado Denver.

Western immunoblotting. After metformin treatment of both cells lines (5–20 mM) for 24 and 48 h, the cells were given three washings with ice-cold PBS. Non-denaturing lysis buffer was then used to prepare the cell lysates as described in reference 57. In order to ensure equal protein loading, the concentration of protein in each sample was determined by the Lowry protein assay using a Bio-Rad DC Protein Assay Kit. The cell lysates were loaded onto 6, 8, 12 and 16% Tris-glycine gels by sodium dodecyl sulfate PAGE (SDS-PAGE). After the proteins were run through the gel, they were transferred onto a nitrocellulose membrane by western blotting. The membranes were then blocked in 5% milk for an hour and then probed with the appropriate primary antibody at 4°C overnight. Afterwards, the corresponding secondary antibodies were then probed for an hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system (GE Healthcare Life Sciences).

Cycloheximide experiment to determine protein stability.

Both cell lines were plated to approximately 60% confluency, and then treated with 50 µg/mL cycloheximide, or a combination of 50 µg/mL cycloheximide and 20 mM metformin, for 0, 3, 6, 9 and 12 h. At the end of the treatment times, cell lysates were prepared and analyzed using the western immunoblotting described above.

Statistical analysis.

Data was calculated and analyzed using SigmaStat 3.5 (Jandel Scientific Software). Student's t-test or one-way ANOVA (analysis of variance between groups) were used to determine significant differences between the treatment and control. Bands produced by western blotting were scanned into Adobe Photoshop 6.0 (Adobe Systems Inc.).

Acknowledgments

This work was supported by NCI R01 grants CA102514 and CA140368.

Abbreviations

AMPK

AMP-activated protein kinase

Cdk

cyclin-dependent kinase

CDKI

cyclin-dependent kinase inhibitor

HPV

human papilloma virus

HNSCC

head and neck squamous cell carcinoma

mTOR

mammalian target of rapamycin

Disclosure of Potential Conflicts of Interest

The authors declare that there are no conflicts of interest.

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