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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2012 Dec 27;139(4):625–633. doi: 10.1007/s00432-012-1366-0

Expression of vascular endothelial growth factor and transcription factors HIF-1, NF-kB expression in squamous cell carcinoma of head and neck; association with proteasome and calpain activities

Liudmila V Spirina 1,✉, Irina V Kondakova 1, Evgeny L Choynzonov 1, Svetlana Y Chigevskaya 1, Dmitry A Shishkin 1, Denis Y Kulbakin 1
PMCID: PMC11824787  PMID: 23269488

Abstract

Introduction

The transcription factors NF-kB, HIF-1 and vascular endothelial growth factors (VEGF) are known to play an important role in pathogenesis of squamous cell carcinoma of head and neck (SCCHN).

Purpose

The aim of the study was to determine the NF-kB, HIF-1 and VEGF, expression their characteristics in squamous cell carcinoma of head and neck.

Methods

Transcription factors and VEGF expression were measured by ELISA kits. Proteasome and calpain activity were determined using specific fluorogenic substrate. Proteasome subunits composition was measured by Western blot analysis.

Results

In the present study, we revealed the connection between SCCHN lymphogenous metastasis development and NF-kB p50 expression. An increase in total, 26S and 20S proteasome activities and calpain activity was observed in cancer tissues in comparison with agreed standard (non-transformed tissue). The dynamics of changes in proteasome activity and proteasome subunits content during lymph nodes metastasis development had a complex pattern. Nonparametric analysis of variance showed the connection between the extent of metastatic affection of regional lymph nodes, total proteasome activity and LMP2 expression. Proteasome and calpain systems corresponded and interacted with each other. We also revealed a positive correlation between the NF-kB p65 and p50 expression and proteasome activity.

Conclusion

Taken together, our results suggest that above mentioned transcription factors and intracellular proteolytic systems are involved in SCCHN progression and metastasis. Moreover, the opportunity of transcription factors regulation by proteasome takes place in oncogenesis of SCCHN. The results provide a basis for new prognostic tests and development of novel targeted therapy.

Keywords: Transcription factors NF-kB p65 and p50, HIF-1, VEGF, Proteasome, Calpain, Squamous cell carcinoma of head and neck

Introduction

Squamous cell carcinoma of the head and neck (SCCHN) is the most common worldwide epithelial malignancy arising in the upper aerodigestive tract, encompassing the oral cavity, oropharynx, hypopharynx, pharynx and larynx. SCCHN is a diverse group of uncommon tumors that frequently are aggressive in their biological behavior with fatal evolution within a few months. Most patients with head and neck cancer have metastatic disease at the time of diagnosis (regional nodal involvement in 43 % and distant metastasis in 10 %) (Ridge et al. 2011). It is known that tumor growth and progression are dependent on the biological characteristics of cancers and on the main regulatory events including expression of the growth and transcription factors. (Spirina et al. 2012a, b; An and Rettig 2005, 2007).

Transcription factor HIF-1 is a heterodimer of alpha- and beta-subunits. Βeta-subunit is constitutionally expressed, whereas the activity of HIF-1 depends on alpha-subunit expression and its posttranslational modifications. Hydroxylation of proline and asparagine residues in HIF-1α results in its binding to the von Hippel–Lindau protein (pVHL), which is followed by HIF-1α polyubiquitination and degradation in the proteasome (Zhou et al. 2006; Klatte et al. 2007). Under hypoxic conditions, stabilized HIF-1α subunits heterodimerize with β subunits to transactivate target genes after nuclear translocation. The result of HIF activation is production of vascular endothelial growth factor (VEGF) (Linder et al. 1998) that stimulates vasculogenesis and angiogenesis.

The key transcription factor is NF-kB that controls the transcription of DNA. NF-kB is found in almost all animal cell types and is involved in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, apoptosis and immune reaction (Baldwin 1996). A subfamily of NF-kB proteins includes five genes encoding five main proteins: NF-kB1, NF-kB2, RelA (p65), RelB and c-Rel. The NF-kB1 and NF-kB2 proteins are synthesized as large precursors, p105, and p100, which undergo processing to generate the mature NF-kB subunits, p50 and p52, respectively. The processing of p105 and p100 is mediated by the ubiquitin/proteasome pathway (Goldberg 2007). The proteins form the homo- and heterodimers of different structure. The active form of transcription factor NF-kB is the heterodimer p65/p50 (Kostadinova et al. 2005; Marui et al. 2005; Juvekar et al. 2011). The ratio of functional NF-kB subunit dimers comprised of p50/p50 homodimers relative to p65/p50 heterodimers can act as a molecular switch in cell processes (Conner et al. 2010). The transcription factors mutually influence each other, and this fact implies the existence of a complex regulation of their expression and activity and a cross-talk between these factors. So far, HIF-1α level has been shown to change in NF-kB-dependent manner (van Uden et al. 2008).

At present, growth of primary tumors and formation of metastases of SCCNH is associated with expression of transcription and growth factors. Increase in HIF-1 expression in SCCHN is combined to microvessel density and involvement of lymph node (Liang et al. 2008). High NF-kB expression was also observed in squamous carcinoma of the larynx and was associated with tumor grade (Jiang et al. 2011). At the same time, use of proteasome inhibitors in squamous cell line leads to the decrease in proangiogenic proteins as well as VEGF (Yan et al. 2010).

The proteolysis is one of the mechanisms for regulation of transcription factors NF-kB and HIF-1α, and the proteasome and calpain systems are the most possible factors to be involved. The proteasome is the multicatalytic complex that consists of the catalytic core (20S) with one or two regulatory particles attached to it. In case when at least one of these particles is PA700 (19S regulatory particle), it is the 26S proteasome that performs mostly ATP- and ubiquitin-dependent proteolysis of multiple cellular proteins. If it is another particle (PA28, PA200) attached, then the proteasome is an activated 20S form, and it is responsible for the cleavage of small, misfolded and short-lived proteins (Kelvin and Reshma 2006). Both the 20S proteasome pool and the 26S proteasome pool may be divided into two large groups of immune and constitutive forms that consist either of constitutive (α1α2α3α4α5α6α7 and β1β2β3β4β5β6β7) or immune (LMP2, LMP7, MECL-1) subunits, respectively (Sorokin et al. 2009; Almond and Cohen 2002). The immune proteasomes contain catalytic subunits LMP7 (β5i), LMP2 (β1i) and MECL1 (β2i) instead of catalytic subunits X (β5), Y (β1) and Z (β2) of constitutive proteasomes. The substitution of constitutive β-subunits by the immune ones in the proteasome is associated with changes in its specificity due to which immune peptides production is performed for their presentation by major histocompatibility complex. It is necessary to note that it is observed the role of proteasome in oncogenesis of breast (Chen et al. 2006), colon (Voutsadakis 2007) and so on. But in case of SCCHN the investigations were performed in cell culture (Chen et al. 2008; Li et al. 2008). High proteasome activity is studied in SCCHN development accompanied by proteasome subunits changes (Spirina et al. 2010).

Calpains belong to the calcium-dependent proteases (Goll et al. 2003). Calpain-1 and calpain-2 are the most known of them that wide spread in human tissues. An increase in calpains expression was detected in squamous cell carcinomas and basal cell carcinomas (BCC) of human skin (Reichart et al. 2003).

The question about proteolytic regulation of growth and transcription factors expression is still open. Decrease in HIF-1α degradation in presence of proteasome inhibitors or under hypoxia leads to a significant increase in both VEGF and its mRNA expression in tumor cells (Molitoris et al. 2009; Yue et al. 2011). Moreover, calpains were shown to be involved in HIF-1α destruction (Zhou et al. 2006). Activation of the NF-kB is effected by proteasomes. NF-kB is presented in cytoplasm in complex with its own repressor IkB. The key moment of NF-kB activation belongs to breaking the ties between the transcription factor and repressor. The IkB inhibitor molecules are modified by a process called ubiquitination, which then leads them to be degraded by proteasomes (Goldberg 2007; Juvekar et al. 2011). Currently, even greater significance is attached to the investigation on the participation of calpains in IkB destruction (Li et al. 2010). The additional mechanism in NF-kB regulation serves the NF-kB protein forming from precursors which is mediated by proteasomes through the modification of p105 (Moorty et al. 2006).

The proteolytic systems are closely linked with each other. It is known that the rise of proteasome activity leads to the calpains activation. The one-way changes in proteolytic activity and expression proteins have been shown the functional division. In opposite to proteasome, the proteolysis performing by calpains is incomplete (Sorimachi et al. 2011). On the other hand, calpain activation leads to an enhanced proteasome activity and is followed by increase in proteasome-dependent protein degradation (Smith and Dodd 2007).

The regulation of growth factors, NF-kB and HIF-1 expression by proteasomes and calpains in human malignancies with their cross-talk has not been clarified yet. In particularly, there is no comprehension of proteolytic systems features and their regulation of transcription and growth factors in cancer metastasis development. Therefore, the aim of our study was to determine the activity of proteasome and calpains on SCCHN, to find their connection with cancer development and to reveal their association with VEGF, HIF-1α and NF-kB expression.

Materials and methods

Patients

Tissues of SCCHN were obtained from 66 patients including 25 patients with cancer of larynx, 23 patients with oral tumors and 18 patients with tongue cancer. According to the ESMO recommendations (TNM, 2009), all patients had squamous cell carcinoma of head and neck T2-3N0-2M0 and underwent surgery at Cancer Research Institute of Siberian Branch of Russian Academy of Medical Sciences, Tomsk, Russian Federation, from January 2008 to January 2011 (mean age 57.1 ± 1.7 years). Radical neck dissection was also used to resect some or all of the cervical lymph nodes to prevent further spread of the disease. The study was approved by the Local Committee for Medical Ethics, and all patients provided written informed consent. Specimens were reviewed by two pathologists separately.

Investigation of the proteasome was performed in samples of cancer tissue and non-transformed tissue, obtained at a distance of not less than 2 cm from the tumor border. The frozen samples were stored at −80 °C.

Preparing tissue homogenates

Tissue samples (100 mg) were homogenized and then resuspended in 300 μL of 50 mM Tris–HCl buffer (pH = 7.5) containing 2 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA and 100 mM NaCl. The homogenate was centrifuged at 10,000×g for 60 min at 4 °C.

Preparing nuclear extract for HIF-1α and NF-kB (p50 and p65) determination

The pellets left after preparing tissue homogenates were resuspended in 50 μL of 50 mM Tris–HCl buffer (pH = 7.5) containing 2 mM ATP, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA and 100 mM NaCl and then centrifuged at 14,000×g for 10 min at 4 °C.

VEGF, HIF-1α and NF-kB (p65 and p50) determination

HIF1-α expression was measured with Caymanchem ELISA kits (USA) in Anthos 2020 ELISA-microplate reader (Biochrom, UK). Nuclear extracts were prepared and purified according to manufacturer’s instructions. Protein concentration in homogenates and nuclear extracts was determined by Lowry. Results of VEGF level measurements were expressed as pg per mg protein, while HIF-1α and NF-kB (p65 and p50) levels were expressed as relative light units per mg protein in well.

Proteasome fractionation

All procedures were carried out on ice or at 4 °C. Proteins from tissue homogenates were fractionated with stepwise concentrations of ammonium sulfate. 26S proteasome-rich fraction was isolated by adding ammonium sulfate to 40 % final concentration, while 20S proteasome-rich fraction was isolated by adding ammonium sulfate to 70 % concentration (Abramova et al. 2004). The fractions were assayed for the proteasome activity.

Proteasome activity assay

Chymotrypsin-like activity of the total, 26S and 20S proteasome pools was measured in cancer and non-transformed tissue homogenates, and in the proteasome fractions, using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin (Suc-LLVY-AMC) in a Hitachi-850 (Japan) fluorimeter at an excitation wavelength of 380 nm and an emission of 440 nm. This substrate is preferentially hydrolyzed by the chymotrypsin-like peptidase activity of the 20S proteasome (Ben-Shahar et al. 1999). The 20S proteasome activity solution contained 20 mM Tris–HCl (pH = 7.5), 1 mM dithiothreitol and 30 μM Suc-LLVY-AMC. The 26S proteasome activity solution additionally contained 5 mM MgCl2 and 1 mM ATP. The reaction was carried out for 20 min at 37 °C and then was stopped by the addition of 1 % sodium dodecyl sulfate. We used the proteasome inhibitor MG-132 to estimate the influence of other proteases. The unit of activity per mg protein was calculated. Protein concentration was determined by Lowry.

Calpains activity assay

The calpains activity was performed in tissue homogenates using the fluorogenic substrate N-Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin (Suc-LLVY-AMC) in a Hitachi-850 (Japan) fluorimeter at an excitation wavelength of 380 nm and an emission of 440 nm (Sandmann et al. 2002).

The calpains activity solution contained 100 mM Tris–HCl (pH = 7.3), 145 mM NaCl and 30 μM Suc-LLVY-AMC. Incubations were performed at room temperature for 30 min in absence or presence of 10 mM CaCl2 and N-Acetyl-L-leucyl-L-leucyl-L-norleucinal (calpain inhibitor I). The reaction was stopped by the addition of 1 % sodium dodecyl sulfate. Calpains activity was measured as fluorogenic units per mg protein. Protein concentration was determined by Lowry.

Electrophoresis

SDS-PAGE was used, according to the method of Laemmli (Laemmi 1970). The samples were incubated for 5–10 min in 62.5 mM Tris–HCl buffer (pH 6.8), containing 2.0 % (w/v) SDS, 5.0 % (v/v)/3-mercaptoethanol, 10 % (v/v) glycerol and 0.0012 % Bromophenol blue.

Western blot analysis

After SDS-PAGE, the gels were allowed to equilibrate for 10 min in 25 mM Tris and 192 mM glycine in 20 % (v/v) methanol. The protein was transferred to 0.2-/xm pore-sized nitrocellulose (GE Healthcare, UK), either at 150 mA or 100 V for 1 h by using a Bio-Rad Mini Trans-Blot electrophoresis cell according to the method described in the manual accompanying the unit. Before incubation with antibodies, the nitrocellulose was incubated in 10 mM Tris-HC1 buffer (pH 7.5), containing 150 mM NaC1 and 0.1 % (v/v) tween-20 for 2 h. The nitrocellulose was incubated in a 1:2,500 dilution of monoclonal mouse anti-human α1α2α3α5α6α7, LMP7(Santa Cruz, USA), Rpt6 (Enzo Life Science, USA) and of polyclonal rabbit anti-human LMP2, PA28β (Santa Cruz, USA) at 20 °C for 1 h, followed by three consecutive washes in the 10 mM Tris-HC1 buffer (pH 7.5), containing 150 mM NaCI (10 min/wash). The nitrocellulose was incubated in a 1:10,000 dilution of anti-mouse or anti-rabbit antibodies for 1 h. After three more 10-min washes, the nitrocellulose samples were incubated in Amersham ECL western blotting detection analysis system according to the method described in the manual accompanying the unit and then were exposed to ECL-films (Amersham, USA). The films were scanned, and graphs of lanes were analyzed in “Image J” computer program. Relative amounts (percentages) of the peak areas were assessed by cutting the peaks and weighing them on an analytical balance. The results were standardized using the β-actin expression in a sample and were expressed in percentages to the proteasome subunits content in non-transformed tissues. The expression of proteasome subunit in normal non-altered tissue was indicated as 100 %.

Statistical analysis

Statistical analysis was performed using Statistica 6.0 software. Normally distributed data are expressed as mean ± SED; otherwise, data are expressed as median (interquartile ranges). To evaluate the difference, either the Student’s t test or the Mann–Whitney test was applied. The Kruskal–Wallis one-way analysis of variance and median test were used for comparing two or more independent samples. Correlation analysis on data was carried out with Spearman rank correlation test. The level of significance was set at p < 0.05.

Results and discussion

HIF-1α, NF-kB and VEGF expression in cancer of head and neck

The expressions of HIF-1α, NF-kB and VEGF in SCCHN are shown in Fig. 1. The results have been estimated the prevalence of active forms of transcription factor NF-kB. The ratio of NF-kB p65 relative to NF-kB p50 less then 1.0 is the sign of non-active dimmers of NF-kB (Goldberg 2007). Thus, the coefficient p65/p50 NF-kB was 1.5 in cancers of head and neck. That was the sign of NF-kB activation. HIF-1α expression was 9.0 (6.2–19.8) RLU per mg protein in well, and VEGF content was 81.8 (62.2–96.6) pg/per mg protein.

Fig. 1.

Fig. 1

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NF-kB p50, p65 and HIF-1α expression in squamous cell carcinoma of head and neck. The results have been estimated the prevalence of active forms of transcription factor NF-kB. Coefficient p65/p50 NF-kB was 1.5 in cancers of head and neck that was the sign of NF-kB activation. HIF-1α expression was 9.0 (6.2–19.8) RLU per mg protein in well

The next step of research was to obtain of transcription and VEGF expression in patients with absence or presence of regional lymph nodes affection (Table 1). The expression of NF-kB was different in metastatic and non-metastatic cancers; interestingly, this dependence had a complex wave-like pattern. The NF-kB p50 expression was enhanced in 2.9-fold in patients with stage T2-3N1 in comparison with the stage T2-3N0. The rise of the affected lymph node amount was conducted with decrease in NF-kB p50 expression: The NF-kB p50 expression was less in T2-3N2 patients in comparison with T2-3N1 group. NF-kB p65 expression was revealed the trends to rise in patients with regional lymph node metastasis.

Table 1.

Tissue levels of transcription factors and VEGF in non-metastatic and metastatic squamous cell carcinoma of head and neck

Groups
T2-3N0, n = 11 T2-3N1, n = 6 T2-3N2, n = 6
Transcription factors expression, RLU per protein in well
 NF-kB p50 6.6 (2.2–17.0) 19.7 (11.8–30.8)* 5.2 (2.8–7.8)**
 NF-kB p65 9.2 (5.9–14.4) 12.5 (10.7–16.7) 8.7 (7.0–19.4)
 NF-kB p65/p50 1.6 (0.7–3.3) 0.6 (0.5–0.8) 2.5 (1.7–3.2)**
 HIF-1α 7.8 (4.6–21.1) 10.9 (10.8–16.7) 9.0 (6.5–14.6)
VEGF expression, pg/per mg of protein 71.1 (48.2–87.7) 92.1 (84.5–98.7) 74.5 (65.2–88.8)
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The results represent the Me (Q1-Q3)

* In comparison with T2-3N0 group p < 0.05; ** In comparison with T2-3N1 group

Wave-like changes was also determined for coefficient p65/p50 NF-kB. Our data indicated the ratio of NF-kB p65 relative to NF-kB p50 has been decrease in patients with stage T2-3N1 in comparison with the stage T2-3N0, and its amount was 0.6. Coefficient p65/p50 NF-kB achieved the level of 2.5 in patients with T2-3N2 stage. The HIF-1 expression did not depend on the cancer progression. However, VEGF expression has been shown the trend to increase with cancer expansion to lymph nodes.

In whole, our results are accorded with the data in which transcription and growth factors expression considered to be main cancer progression markers of SCCHN (Liang et al. 2008). The changes of NF-kB p50 expression were very important for cancer metastasis development. Probably, it should be the result of enhanced proteasome activity (Moorty et al. 2006). To confirm the connection between the transcription and VEGF expression and cellular proteolysis the proteasomes activity, content of its constitutive and immune subunits and calpain activity in SCCHN was investigated.

Proteasome and calpain activities increase in cancer of head and neck

The total, 26S and 20S proteasome activities has been found to be higher in squamous cell carcinomas of the head and neck than in non-transformed tissues (Fig. 2). It is known that the proteasome activity is associated with its subunits content. (Almond and Cohen 2002; Arlt et al. 2009). Expression of total proteasome pool, that is represented the rate of α1α2α3α5α6α7 proteasome subunits, was lower in cancer tissues from their level in non-transformed tissue (agreed standard) (p < 0.05). At the same time, the expression of immune proteasome subunits and regulatory subunit rpt6 was higher in cancer tissues. The level of LMP7, LMP2, PA28β and rpt6 proteasome subunits was increased in 60, 47, 22 and 51 % in tumor tissues in comparison with normal one (Fig. 3). Proteasome activation was accompanied with growth of calpain activity. It was revealed that the total calpain activity in cancers was increased in 1.7-fold in comparison with non-transformed tissue.

Fig. 2.

Fig. 2

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Total, 26S and 20S proteasome activities and calpain activity in squamous cell carcinoma of head and neck. The results represent the Me (Q1-Q3); * in comparison with normal tissue p < 0.05. Proteasome activation was accompanied with growth of calpains activity. It was detected the high total, 26S and 20S activities

Fig. 3.

Fig. 3

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Proteasome subunits content in squamous cell carcinoma of head and neck. a The expression of proteasome subunit in normal non-altered tissue was indicated as 100 %. It was revealed that the low content of α1α2α3α5α6α7 proteasome subunit expression. The expressions of immune proteasome subunits and regulatory subunits rpt6 and PA28 were higher in cancer tissues. * in comparison with normal tissue p < 0.05; the results were standardized using the β-actin expression in a sample and were expressed in percentages to the proteasome subunits content in non-transformed tissues. b Western blotting analysis has been shown the expression of proteasome subunits in cancer and non-transformed tissues: 1, 3—western blots of cancer tissues; 2, 4—western blots of non-transformed tissues

Connection between protease activities and clinico-morphological parameters in SCCHN was studied. It was found twofold increase in total proteasome activity higher in cancer tissue with T3 stage in comparison with T2 stage. It was also observed the growth of calpain activity in 14-fold in tumors with size T3 in comparison with tumor size T2.

Activities of proteasomes, their pools in tumor tissue seemed to be depended on the stage of lymphogenous metastasis (N) (Table 2). The total proteasome activity in the tumor tissues obtained from patients with T2-3N2 stage was increased in 1.4-fold in comparison with patients with T2-3N1 stage, which was accompanied with decrease in LMP2 expression in 1.2-fold. The rate of LMP2 in proteasome core is depended on the level of ubiquitinated proteins and is connected with immune response dysfunction and with defects of histocompatibility complex 1 class presentation (Hensley et al. 2010). The activity of other cellular enzymes—calpains—was increased in 1.4- and 1.7-fold in group of patients with T2-3N1 and T2-3N2 stages in comparison with non-metastatic ones.

Table 2.

Proteasome activity, their subunit content and calpains activity in non-metastatic and metastatic squamous cell carcinoma of head and neck

Groups
T2-3N0, n = 36 T2-3N1, n = 20 T2-3N2, n = 10
Proteasome activity, 1,000 ME/per mg protein
 Total activity 58.1 (43.1–117.1) 43.5 (31.5–68.0) 94.4 (60.0–268.3)**
 26S 20.0 (14.8–38.0) 21.6 (13.3–35.9) 34.0 (11.1–47.8)
 20S 45.9 (35.5–87.0) 35.9 (17.5–62.5) 45.0 (33.3–87.0)
Proteasome subunit content in cancer tissue in  % to their content in non-transformed one.
 α1α2α3 α5α6α7 83.5 (58.3–91.8) 81.0 (56.3–96.0) 62.3 (10.0–81.7)
 LMP7 138.6 (89.0–211.9) 184.1 (116.6–276.9) 172.8 (84.2–300.0)
 LMP2 143.1 (105.4–167.1) 154.5 (11.3–194.3) 110.1 (81.1–138.5)**
 PA28β 128.5 (105.4–168.3) 120.1 (104.2–163.9) 116.0 (98.2–180.0)
 rpt6 151.5 (101.0–222.8) 128.6 (95.0–194.4) 157.9 (112.0–305.3)
Calpains activity, 1,000 ME/per mg protein 119.0 (66.2–245.2) 142.4 (56.6–345.0)* 204.7 (74.0–335.5)**
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The results represent the Me (Q1-Q3)

* In comparison with T2-3N0 group p < 0.05; ** In comparison with T2-3N1 group

Changes in proteasome activity, transcription factors and VEGF expression in cancer expansion and lymph nodes metastasis development had the opposite trend; the maximal high proteasome activity in T2-3N2 stage was conducted with minimal content of transcription factors. That was the sign of their proteasomal degradation. In common, the movements of proteasome subunit changes in cancer metastasis development were different. But it was found the decrease in LMP2 level, and transcription factors expression had the same relation in metastatic cancers. It was also observed the association of lymphogenous metastasis with the transcription factors, VEGF expression, proteasome and calpain activity using the nonparametric analysis of variance. Kruskal–Wallis ranking test was revealed the connection of cancer stage N with the NF-kB p50 expression (p1 = 0.05) and with total proteasome activity (p1 = 0.05). Median test was found out the tie of cancer metastasis development with rate of LMP2 expression (p2 = 0.05). The performed analysis confirmed the involvement of transcription factors and cellular proteolysis in cancer expansion to lymph nodes.

Correlations between the proteasome activity, transcription factors and VEGF expression were found in SCCHN

Positive relationships between activities of all of the proteasome pools in endometrial tumors were found. We observed statistically significant correlations between the 20S and 26S proteasome activities (r = 0.37; p < 0.05), between the total proteasome and 20S activities (r = 0.74; p < 0.05) and between the total and 26S proteasome activities (r = 0.49; p < 0.05). Proteasome activation was corresponded with rise of calpains activity. Positive correlations were found between the total proteasome activity, 26S proteasome activity and activity of calpains (r1 = 0.7, p = 0.02; r2 = 0.7, p = 0.01).

The analysis of data (Fig. 4) has been shown the relationships between the total proteasome activity and NF-kB p65 expression (r = 0.6; p = 0.001) and between the 20S proteasome activity and NF-kB p50 and p65 expression (r1 = 0.58, p = 0.04; r2 = 0.45; p < 0.05). It was also observed the association of HIF-1α expression with VEGF expression (r = 0.4; p = 0.04) in cancer. In Goldberg A.L. study (Goldberg 2007), it was revealed the influence of transcription factors NF-kB on HIF-1α expression. Our data have been shown the connection of HIF-1α expression with NF-kB p50 expression (r = 0.65; p = 0.0001) and NF-kB p65 expression (r = 0.62; p = 0.002) in SCCHN. Besides it was found dependence of VEGF expression on NF-kB p50 level (r = 0.4; p = 0.036). Probably this fact was mediated through HIF-1 expression changes.

Fig. 4.

Fig. 4

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Scatterplots of the 20S proteasome activity against the NF-kB p50 expression (a), NF-kB p65 expression (b) in SCCHN; Scatterplots of the HIF-1α expression against the NF-kB p50 (c) and NF-kB p65 expression (d) in SCCHN. The analysis of data has been shown the relationships between the 20S proteasome activity and NF-kB p50 and p65 expression (r1 = 0.58, p = 0.04; r2 = 0.45; p < 0.05). Connection of HIF-1α expression with NF-kB p50 expression (r = 0.65; p = 0.0001) and NF-kB p65 expression (r = 0.62; p = 0.002) have been found in SCCHN

Cross-talk between the expression of transcription factors, VEGF, proteasome and calpains activity in SCCHN

The hypothetic schema of regulation of growth and transcription factors by proteasomes and calpains is presented on Fig. 5. Proteasome and calpains systems corresponded and interacted with each other in cancer tissue. It was necessary to estimate the role of calpains in transcription factors, and VEGF expression in cancer of head and neck did not find out. Proteasome activation had the prevalence in SCCHN progression through existence of multiple controls of transcription and growth factors expression. It is known that the degradation of IkB proceeding in proteasomes is the key moment in NF-kB activation (Juvekar et al. 2011). The main importance in NF-kB regulation belongs to posttranslational modification of NF-kB precursor performed by proteasomes (Moorty et al. 2006). As a result of the conducted research relationships between the NF-kB p65, NF-kB p50 expression and proteasome activity in cancers of head and neck have been found.

Fig. 5.

Fig. 5

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Cross-talk between the proteasome activities and expression of transcription and growth factors in SCCHN—positive relationship. The schema of regulation of growth and transcription factors by proteasomes and calpains in SCCHN is hypothetic. Proteasome and calpain systems corresponded and interacted with each other in cancer tissue. Proteasome activation takes part in SCCHN progression through existence of multiple controls of transcription and growth factors expressions. As a result of the conducted research relationships between the NF-kB p65, NF-kB p50 expressions and proteasome activity in cancers of head and neck have been found. Angiogenesis is the main process that could determine cancer progression. Association between the HIF-1α expression and proteasome activity was not revealed in SCCHN. Considering the presence of relationships between the NF-kB and HIF-1 expressions being under the control of proteasomes, we suppose angiogenesis regulation in cancers of head and neck is performed by NF-kB

Angiogenesis is the main process that could determine cancer progression. It is known that the development of new vessels in tumor is linked to the HIF-1 expression which is degradated by proteasome. The association between the HIF-1α expression and proteasome activity was not revealed in SCCHN. Considering the presence of relationships between the NF-kB and HIF-1 expression being under the control of proteasomes, we suppose angiogenesis regulation in cancers of head and neck is performed by NF-kB.

Conclusion

The results of our investigation allowed to find the connection between the proteasome activity and transcription factors expression in cancers of head and neck. As the result of investigation, the hypothetic schema of proteolytic regulation of transcription factors and VEGF expression in squamous cell carcinoma of head and neck was obtained. It was revealed that the activation of cellular proteolytic systems and changes of their subunits contents in cancer progression and metastasis development. The revealed activation of intracellular proteolytic systems and changes in the proteasome subunits content in cancer progression and metastasis development was mainly associated with NF- kB expression. Additional studies, in vitro and in vivo, will help to elucidate the contribution of proteolytic systems to therapy and clinical outcome of patients with of SCCHN as well as in the target therapy development.

Acknowledgments

This study was supported by the Federal target program “Scientific and scientific-pedagogical personnel of innovative Russia” in 2009–2013 (Governmental Contract No. P320).

References

  1. Abramova EB, Astakhova TM, Erokhov PA, Sharova NP (2004) Multiple forms of the proteasomes and some approaches to their separation. Izv Akad Nauk Ser Biol 2:150–156 [PubMed] [Google Scholar]
  2. Almond JB, Cohen GM (2002) The proteasome: a novel target for cancer chemotherapy. Leukemia 16(4):433–443 [DOI] [PubMed] [Google Scholar]
  3. An J, Rettig MB (2005) Mechanism of von Hippel-Lindau protein-mediated suppression of nuclear factor kappa B activity. Mol Cell Biol 25:7546–7556 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. An J, Rettig MB (2007) Epidermal growth factor receptor inhibition sensitizes renal cell carcinoma cells to the cytotoxic effects of bortezomib. Mol Cancer Ther 6(1):61–69 [DOI] [PubMed] [Google Scholar]
  5. Arlt A, Bauer I, Shagmayer C, Telep J et al (2009) Increased proteasome subunit protein expression and proteasome activity in colon cancer relate to an enhanced activation of nuclear factor E2-related factor 2 (Nrf2). Oncogene 28(45):3983–3996 [DOI] [PubMed] [Google Scholar]
  6. Baldwin AS (1996) The NF-kappa B and I-kappa B proteins: new discoveries and insights. Annu Rev Immunol 14:649–683 [DOI] [PubMed] [Google Scholar]
  7. Ben-Shahar S, Komlosh A, Nadav E et al (1999) 26S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate. The J of Biol Chem 274(31):21963–21972 [DOI] [PubMed] [Google Scholar]
  8. Chen C, Seth AK, Aplin AE (2006) Genetic and expression aberrations of E3 ubiquitin ligases in human breast cancer. Mol Cancer Res 4:695–707 [DOI] [PubMed] [Google Scholar]
  9. Chen Z, Rickel JL, Malhotra PS, Nottingham L, Bagain L, Lee TL, Yen NT, Van Waes C (2008) Differential bortezomib sensitivity in head and neck cancer lines corresponds to proteasome, nuclear factor-kappaB and activator protein-1 related mechanisms. Mol Cancer Ther 7(7):1949–1960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Conner JR, Smirnova II, Moseman AP, Poltorak A (2010) IRAK1BP1 inhibits inflammation by promoting nuclear translocation of NF-kappaB p50. Proc Natl Acad Sci USA 107(25):11477–11482 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldberg AL (2007) Functions of the proteasome: from protein degradation and immune surveillance to cancer therapy. Biochem Soc Trans 35:12–17 [DOI] [PubMed] [Google Scholar]
  12. Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83:731–801 [DOI] [PubMed] [Google Scholar]
  13. Hensley SE, Zanker D, Dolan BP, David A, Hickman HD, Embry AC, Skon CN, Grebe KM, Griffin TA, Chen W, Bennik JR, Yewdell JW (2010) Unexpected role for the immunoproteasome subunit LMP2 in antiviral humoral and innate immune responses. J Immunol 184(8):4115–4122 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jiang LZ, Wang P, Deng B, Huang C, Tang WX, Lu HY, Chen HY (2011) Overexpression of forkhead box M1 transcription factor and nuclear factor-kB in laryngeal squamous cell carcinoma: a potential indicator for poor prognosis. Hum Pathol 42(8):1185–1193 [DOI] [PubMed] [Google Scholar]
  15. Juvekar A, Manna S, Ramaswami S, Chang TP, Vu HY, Ghosh CC, Celiker MY, Vancurova I (2011) Bortezomib induces nuclear translocation of IkBα resulting in gene-specific suppression of NF-kB-dependent transcription and induction of apoptosis in CTCL. Mol Cancer Res 9(2):183–194 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kelvin JAD, Reshma S (2006) Preferential degradation of oxidized proteins by the 20S proteasome may be inhibited in aging and in inflammatory neuromuscular diseases. Neurology 66:93–96 [DOI] [PubMed] [Google Scholar]
  17. Klatte T, Seligson DB, Riggs SB, Leppert JT, Berkman MK (2007) Hypoxia-inducible factor 1 alpha in clear cell renal cell carcinoma. Clin Cancer Res 13:7388–7393 [DOI] [PubMed] [Google Scholar]
  18. Kostadinova RM, Nawrocki AR, Frey FJ, Frey BM (2005) Tumor necrosis factor alpha and phorbol 12-myristate-13-acetate down-regulate human 11beta-hydroxysteroid dehydrogenase type 2 through p50/p50 NF-kappaB homodimers and Egr-1. FASEB J 19(6):650–652 [DOI] [PubMed] [Google Scholar]
  19. Laemmi UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–685 [DOI] [PubMed] [Google Scholar]
  20. Li C, Li R, Grandis JR, Johnson DE (2008) Bortezomib induces apoptosis via Bim and Bik up-regulation and synergizes with cisplatin in the killing of head and neck squamous cell carcinoma cells. Mol Cancer Ther 7(6):1647–1655 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li C, Chen S, Yue P, Deng X, Lonial S, Khuri FR, Sun SY (2010) Proteasome inhibitor PS-341 (bortezomib) induces calpain-dependent IkappaB(alpha) degradation. J Biol Chem 285(21):16096–16104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liang X, Yang D, Hu J, Hao X, Gao J, Mao Z (2008) Hypoxia inducible factor-1alpha expression correlates with vascular endothelial growth factor-C expression and lymphangiogenesis/angiogenesis in oral squamous cell carcinoma. Anticancer Res 28:1659–1666 [PubMed] [Google Scholar]
  23. Linder C, Linder S, Munck-Wikland E, Auer G, Aspenblad U (1998) Evaluation of tissue and serum VEGF in patients with head and neck carcinoma. Angiogenesis 2:365–372 [DOI] [PubMed] [Google Scholar]
  24. Marui N, Medford RM, Ahmad M (2005) Activation of RelA homodimers by tumor necrosis factor alpha: a possible transcriptional activator in human vascular endothelial cells. Biochem J 390:317–324 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Molitoris KH, Kazi AA, Koos RD (2009) Inhibition of oxygen-induced hypoxia-inducible factor-1alpha degradation unmasks estradiol induction of vascular endothelial growth factor expression in ECC-1 cancer cells in vitro. Endocrinology 150(12):5405–5414 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Moorty AK, Savinova OV, Ho JQ, Wang VY, Vu D, Ghosh G (2006) The 20S proteasome processes NF-kappaB1 p105 into p50 in a translation-independent manner. EMBO J 25(9):1945–1956 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reichart J, Welter C, Mitschele T, Classen U, Meineke V, Seifert M (2003) Different expression patterns of calpain isozymes 1 and 2 (CAPN1 and 2) in squamous cell carcinomas (SCC) and basal cell carcinomas (BCC) of human skin. J Pathol 199(4):509–516 [DOI] [PubMed] [Google Scholar]
  28. Ridge JA, Glisson BS., Lango MN, Feigenberg S (2011) Head and neck tumors cancer management, 14th edn. http://cancernetwork.com/cancer-management/head-and-neck/article/10165/1802498 Accessed 2 November 2011
  29. Sandmann S, Prenzel F, Shaw L, Schauer R, Unger T (2002) Activity profile of calpains I and II in chronically infarcted rat myocardium–influence of the calpain inhibitor CAL 9961. Br J Pharmacol 135(8):1951–1958 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Smith IJ, Dodd SL (2007) Calpain activation causes a proteasome dependent increase in protein degradation and inhibits the Akt signaling pathway in rat diaphragm muscle. Exp Physiol 92(3):561–573 [DOI] [PubMed] [Google Scholar]
  31. Sorimachi H, Hata S, Ono Y (2011) Calpain chronicle—an enzyme family under multidisciplinary characterization. Proc Jpn Acad Ser B Phys Biol Sci 87:287–327 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sorokin AV, Kim ER, Ovchinnikov LP (2009) Proteasome system of protein degradation and processing. Biochemistry (Mosc) 74(13):1411–1442 [DOI] [PubMed] [Google Scholar]
  33. Spirina LV, Kondakova IV, Choinzonov EL, Sharova NP, Chigevskaya SY, Shishkin DA (2010) Activity and subunit composition of proteasomes in head and cervical squamous cell carcinomas. Bull Exp Biol Med 149(1):82–85 [DOI] [PubMed] [Google Scholar]
  34. Spirina LV, Yunusova NV, Kondakova IV, Kolomiets LA, Koval VD, Chernyshova AL, Shpileva OV (2012a) Association of growth factors, HIF-1 and NF-kB expression with proteasomes in endometrial cancer. Mol Biol Rep 39(9):8655–8662 [DOI] [PubMed] [Google Scholar]
  35. Spirina LV, Bochkareva NV, Kondakova IV, Kolomiets LA, Shashova EE, Koval VD, Chernyshova AL, Asadchikova ON (2012b) Regulation of insulin-like growth NF-kappa B proteasome system in endometrial cancer. Mol Biol (Mosk) 46(3):452–460 [PubMed] [Google Scholar]
  36. van Uden P, Kenneth NS, Rocha S (2008) Regulation of hypoxia-inducible factor-1alpha by NF-kappaB. Biochem J 412(3):477–484 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Voutsadakis IA (2007) Pathogenesis of colorectal carcinoma and therapeutic implications: the role of the ubiquitin-proteasome system and Cox-2. J Cell Mol Med 11(2):252–337 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yan M, Xu Q, Zhang P, Zhou X, Chen W (2010) Correlation of NF-kB signal pathway with tumor metastasis of human head and neck squamous cell carcinoma. BMC Cancer 10:437 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Yue CX, Ma J, Zhou HJ (2011) The effect of RhoA and proteasome inhibitor MG132 on angiogenesis in tumors. Sichuan Da Xue Xue Bao Yi Xue Ban 42(4):445–501 [PubMed] [Google Scholar]
  40. Zhou J, Kohl R, Herr B, Frank R, Brune B (2006) Calpain mediates a von Hippel-Lindau protein-independent destruction of hypoxia-inducible factor -1α. Mol Biol Cell 17(4):1549–1558 [DOI] [PMC free article] [PubMed] [Google Scholar]

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