Ginekologia Polska nr 06 2017-5

 

ORIGINAL PAPER / GYNECOLOGY

Potentialization of N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) cytotoxic activity by 2-(1-adamantylamino)-6-methylpyridine (AdAMP) in human ovarian cancer cells

Jacek Sieńko1, Witold Lasek2, Justyna Teliga-Czajkowska3, Roman Smolarczyk4, Krzysztof Czajkowski1

12nd Department of Obstetrics and Gynecology, Medical University of Warsaw, Poland
2Department of Immunology, Centre for Biostructure Research, Medical University of Warsaw, Poland
3Department of Obstetrics and Gynecology Didactics, Medical University of Warsaw, Poland
4Department of Gynecological Endocrinology, Medical University of Warsaw, Poland

Corresponding author:

Jacek Sieńko

2nd Department of Obstetrics and Gynecology, Medical University of Warsaw

Karowa St. 2, 00315 Warsaw, Poland

e-mail: jacek.sienko@wum.edu.pl

ABSTRACT

Objectives: TNF is one of the key cytokines involved in cancer development. TNF signaling can result in both stimulating and inhibitory signals that can result in opposite biological effects in cancerogenesis. 2-(1-adamantylamino)-6-methylpyridine (AdAMP) enhances TNF secretion whereas N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) is a NF-κB inhibitor potentially stimulating proapoptotic TNF signals. The aim of the study was to assess the effect of TPCK in combination with AdAMP on human ovarian cells.

Material and methods: CAOV-1 human ovarian cell line was incubated with TPCK and AdAMP for 24 hours. The cytotoxic effect was evaluated in a crystal violet assay. A monoclonal antibody against TNF, Infliximab, was added to examine the possible mechanism of interactions.

Results: Depending on concentration, AdAMP potentialized cytotoxic activity of TPCK or had a synergistic effect with TPCK. Infliximab did not reverse cytotoxicity of AdAMP and TPCK and in some cytotoxic and non-cytotoxic concentrations even enhanced their cytotoxicity.

Conclusions: AdAMP and TPCK cytotoxicity seems to be dependent on TNF signaling, however, the exact mechanism of interactions remains unclear.

Key words: AdAMP, TPCK, TNF, NF-kB inhibitors, proteasome, ovarian cancer, cell line

Ginekologia Polska 2017; 88, 6: 307311

INTRODUCTION

Ovarian cancer remains the main cause of mortality among malignancies of female reproductive tract in developed countries [1]. Unsatisfactory survival rates are associated with late diagnosis and in consequence with inadequate treatment [2]. Contemporary management of advanced ovarian cancers usually includes cytoreductive surgery combined with adjuvant platinium-taxane based chemotherapy. Although the response rate in the first line treatment is relatively high, relapses account for about 70% of cases [3]. Treatment of recurrent ovarian cancer that is based mainly on various chemotherapy regimen poses a problem of drug resistance, drug toxicity and decreased quality of life what in turn is the limitation of further treatment options and decreases overall survival [4]. Thus, intervention with chemopreventive agents or new adjuvant therapy may offer a desirable option for the improvement in ovarian cancer management [5].

TNF is a multipotent cytokine that plays an important role in cancer growth regulation. The main source of TNF are macrophages and monocytes but this cytokine can also be secreted by other cells including human ovarian cancer cells [6]. A biological effect of TNF is a result of stimula- ting and inhibitory signals that can give opposite results in different conditions. TNF-R1 is involved in most pathways, whereas signaling through TNF-R2 is less understood [7].

The pleiotropic actions of TNF range from proliferative responses such as cell growth and differentiation, through inflammatory effects and the mediation of immune responses, to destructive cellular outcomes such as apoptotic and necrotic cell death mechanisms [8]. Procaspase-8 activation is the essential pathway of TNF induced apoptosis, whereas there are two arms of proliferative signals transducing that ultimately result in the activation of two major transcription factors: NF-κB and c-Jun. The existence of extensive cross talk between apoptosis, NF-κB and c-Jun signaling pathways causes that in the absence of NF-κB activity cellular susceptibility to TNF-induced apoptosis increases, whereas enforced activation of NF-κB protects against apoptosis [9].

Several adamantylamino-pyridine and -pyrimidine derivatives have TNF production-enhancing properties. 2-(1-adamantylamino)-6-methylpyridine (AdAMP) is the most potent of these adamantane derivatives, on some biological functions and TNF production by normal and neoplastic cells [10, 11]. In the previous study we demonstrated a strong TNF production-enhancing activity of AdAMP in human ovarian cancer cell line CAOV1 [12].

Adamantane derivatives were used for treatment of type A Influenza and the Parkinson’s disease [13, 14]. Antiproliferative activity of adamantane derivatives was found against murine leukemia cells (L1210), human T-lymphocyte cells (CEM), and cervix carcinoma cells (HeLa) [15].

NF-kB inhibitors constitute a heterogenic group of agents that can inhibit a proliferative pathway of TNF signaling. The well-known NF-kB inhibitors are some antioxidants, IkBa phosphorylation and/or degradation inhibitors, and proteasome inhibitors, i.e. N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK), pyrrolidine dithiocarbamate or MG-132 [16–18]. Some other agents like non-steroidal anti-inflammatory drugs, glucocorticoids, and cytokines (Il-10) have the activity of NF-kB inhibitors [18–20]. A reversible 26S proteasome inhibitor, bortezomib, that has been recently approved by the Federal Drug Administration (FDA) and the European Regulatory Agency (EMEA) for the treatment of multiple myeloma at least partly acts through the inhibition of NF-κB activity [21].

OBJECTIVES

In this study we tried to investigate the effect of N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) in combination with 2-(1-adamantylamino)-6-methylpyridine (AdAMP) on human ovarian cell line CAOV-1 growth and to explain the possible mechanism of their action.

MATERIAL AND METHODS

Human ovarian cancer cells

The CAOV-1 line was a human ovarian cell line established in Jagiellonian University in Krakow.

The cell line was cultured in Dulbecco’sMEM with 4.5 g/L glucose, sodium pyruvate and Glutamax-1 (high glucose DMEM) supplemented with antibiotic-antimycotic, 50 µM 2-mercaptoethanol and 10% fetal calf serum (FCS) (all from Gibco BRL, Life Technologies, Paisley, UK). Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.

Reagents

2-(1-adamantylamino)-6-methylpyridine (AdAMP) was synthesized at the Institute of Chemistry, Agriculture University of Warsaw, as described previously [22].

N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) was purchased in Sigma (St. Louis, USA) . Both drugs were prepared as 100 mM stock solutions in DMSO and were diluted to the required concentration in culture medium.

Infliximab (Remicade®), a monoclonal antibody against TNF, was obtained from Janssen Biotech, Inc. (Belgium). The stock solution (10 mg/mL) was prepared in distilled water.

Crystal violet assay

The cytotoxic effect of TPCK and/or AdAMP on ovarian cancer cells was tested in a standard crystal violet assay. The dye in this assay, crystal violet, stains DNA of adherent cells in monolayer. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture.

Cells were incubated in 96-well plates (2 × 104/200 μL/well) with AdAMP (final concentrations: 25, 50, 100 and 200 μM) and TPCK (final concentrations: 8, 16, 32 and 64 μM), alone or in combination, for 24 h. In further experiments Infliximab at the concentration of 1, 10 or 100 µg/mL was added to the wells with AdAMP and TPCK and the cells were incubated in the same way. At the end of each incubation medium was removed and each well was washed with 200 µL of phosphate buffered saline (PBS). 50 µL of 0.1% water solution of crystal violet was added to each well for 10 minutes at room temperature. Then the plates were washed in distillated water. Crystals of violet were dissolved by adding 200 µL of 1% solution of sodium dodecyl sulfate (SDS) and shaking the plates. The plates were read on an ELISA reader (SLT-Labinstruments, Salzburg, Austria) using a 550 nm filter.

The means and standard deviations were determined for triplicate samples. The cytotoxic effect was expressed as the relative viability and was calculated as follows: relative viability = [(experimental absorbance - background absorbance)/(absorbance of vehicle-treated cells background absorbance) ×100.

Statistical analysis

Statistical evaluations were performed with Statistica 7.0 software (StatSoft, Poland). Comparisons between groups of continuous outcomes were performed by Student’s t-test after testing for normal distribution by the Kolmogorov-Smirnov test. P < .05 was considered significant.

Chou-Talalay analysis was performed to evaluate the strength of interaction between the drugs [23]. The resulting combination index (CI) was calculated. CI < 0.9 was considered to be synergism. Dose reduction index (DRI) a measure of how many folds the dose of each drug in a synergistic combination may be reduced was calculated using Calcusyn software.

RESULTS

AdAMP and TPCK alone are cytotoxic to CAOV-1

To evaluate the cytotoxic effect of AdAMP and TPCK we incubated CAOV-1 cell line in 96-well plates as described above for 24 hours and assessed cell culture viability in crystal violet assay. AdAMP was not cytotoxic to CAOV-1 line in the concentration of 25, 50 and 100 µM. The cytotoxic effect of AdAMP was statistically significant at the concentration of 200 µM (P < 0.01) (Fig. 1A).

16, 32, 64 and 128 µM TPCK was cytotoxic to CAOV-1 line. TPCK at the concentration of 8 µM had no effect on the cell line viability (Fig. 1B).

70171.png 

Figure 1. Cytotoxic effect of AdAMP (A) and TPCK (B) on CAOV-1 cell line. Values are mean ± SD. *P < 0.05, #P < 0.01, ^P< 0.001

70186.png 

Figure 2. Cytotoxic effect of AdAMP and TPCK on CAOV-1 cell line. Values are mean ± SD. P value presents differences in series between cells non-stimulated and stimulated by AdAMP. *P < 0.001, #P < 0.0001

Combinations of AdAMP and TPCK synergistically inhibit the growth of CAOV-1 cells in vitro

To determine if effectiveness of these two agents could be enhanced by applying them together we tested 25, 50, 100 and 200 µM AdAMP combined with 16, 32, 64 and 128 µM TPCK. AdAMP potentialized cytotoxic activity of TPCK in the following concentrations: 50 µM AdAMP + 64 µM TPCK, 100 µM AdAMP + 3264 µM TPCK, 200 µM AdAMP + 8128 µM TPCK. Only the combination of 200 µM AdAMP and 32 µM TPCK resulted in the synergistic interaction (CI = 0.598) (Fig. 3). Dose Reduction Index (DRI) for that combination of drugs was 1.946 for AdAMP and 11.883 for TPCK.

70221.png 

Figure 3. Cytotoxic interactions of AdAMP and TPCK. CI combination index, CI was calculated from cytotoxic activity of drug combination. CI + 1SD < 0.9 is considered to be synergism

TNF blocking by Infliximab does not reverse the synergistic effect of AdAMP and TPCK

To answer the question if potentialization/synergy in cytotoxic activity of AdAMP and TPCK is the result of enhanced TNF secretion associated with NF-κB pathway inhibition we blocked TNF with Infliximab in the concentration of 1, 10 or 100 mg/L. As Infliximab was cytotoxic to CAOV-1 cell line in the concentration of 100mg/L (relative viability 84.6 ± 4.0%, P < 0.0005) cellular viability was compared between the group incubated only with Infliximab or with Infliximab and AdAMP and TPCK. Infliximab did not reverse cytotoxic activity neither in 200 µM AdAMP and 32 µM TPCK nor in 100 µM AdAMP and 32 µM TPCK. 1, 10, 100 mg/L of Infliximab even enhanced cytotoxicity of 200 µM AdAMP and 32 µM TPCK and 10 mg/L of Infliximab had similar effect when added to 100 µM AdAMP and 32 µM TPCK (Fig. 4).

70205.png 

Figure 4. Influence of Infliximab on combined AdAMP and TPCK cytotoxicity in CAOV-1 cell line. Values are mean ± SD. P value presents differences between cells incubated only with Infliximab or with Infliximab and AdAMP and TPCK. *P < 0.01, #P < 0.001

DISCUSSION

A combination chemotherapy based on adjuvant mechanisms of action of drugs has become the standard approach in the treatment of ovarian cancer. In target therapy the complementary response to different agents usually includes interfering with different stages of cell signaling what in turn results in augmenting or silencing the desired reaction [24].

The study of Pazhang et al. is an example of a combination target therapy with NF-kB inhibitor [25]. The author showed synergistic effects of cytotoxic activity of NF-kB inhibitor, celastrol, and X-linked inhibitor of apoptosis protein (XIAP) inhibitor, embelin used in combination in an acute myeloid leukemia cell line, HL-60. He explained that the synergy of the two agents may be due to cross-talk between NF-κB and XIAP pathway of signal transduction.

In the present study we demonstrated that a NF-kB inhibitor, N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) is an active agent cytotoxic to CAOV-1 ovarian cell line. Its cytotoxic activity can be augmented by 2-(1-adamantylamino)-6-methylpyridine (AdAMP) in some concentrations. It is noteworthy that AdAMP potentialized cytotoxic activity of TPCK not only in the concentration in which AdAMP had cytotoxic activity alone (200 µM) but also in the concentration of 50100 µM in which it was not cytotoxic to CAOV-1 cell line when applied as the only agent. We hypothesized that it could be at least partly due to the enhanced autocrine secretion of TNF associated with NF-kB pathway inhibition. This idea seemed to be supported by our previous study where we demonstrated a stimulation of TNF secretion by CAOV-1 cell line in the concentrations of 0.1 to less than 100 µM [12]. The real potential of CAOV-1 to secrete TNF in higher concentration of AdAMP is unknown as it is confounded by decreased viability of ovarian cancer cells.

The synergistic effect of AdAMP and TPCK defined according to Chou and Talalay [23] was observed only in the combination of 200 AdAMP AdAMP and 32 µM TPCK. However, it is necessary to emphasize that synergy can only be calculated if both drugs have cytotoxic activity. So, the CI had no application in the AdAMP concentrations of less than 200 µM that were “non-toxic” concentrations alone.

The experimental model with Infliximab added to AdAMP and TPCK showed that neither the synergy between AdAMP and TPCK nor the augmentation of cytotoxic activity of TPCK by AdAMP can be entirely explained by TNF-dependent apoptosis. Blocking TNF by Infliximab did not reverse the cytotoxic effect of AdAMP and TPCK what can suggest that TNF-induced-apoptosis with NF-κB pathway inhibition does not have be the dominating mechanism of action in this situation. On the other hand, Infliximab binds both soluble and membranous TNF and activates in vitro antibody- and complement-dependent cellular cytotoxicity by its Fc portion [26]. Lugering et al. showed that TNF inhibition by Infliximab causes apoptosis in T-cells through the activation of caspase-8, -9, -3 and the increased transcription of the pro-apoptotic proteins Bax and Bak [27, 28]. It is not proved that this mechanism of action of Infliximab can be observed in ovarian cancer cells but we are aware that the potential of Infliximab to stimulate apoptosis could conceal the real action of AdAMP and TPCK in our model. In light of these investigations the influence of Infliximab both in cytotoxic and non-cytotoxic concentrations on the viability of CAOV-1 cell line treated with AdAMP and TPCK observed in our study can indirectly suggest that AdAMP and TPCK interact through the TNF signaling pathway. Unfortunately, multidirectional and dose-dependent TNF action makes it difficult to unambiguously prove one specific pathway of TNF signaling [29].

CONCLUSIONS

We conclude that 2-(1-adamantylamino)-6-methylpy- ridine and N-a-tosyl-L-phenylalanine chloromethyl ketone are agents active against ovarian cancer cells and applied together reveal synergy or potentialization of cytotoxic activity. The molecular mechanism of that interactions remains unclear, however, it is probable that they act through TNF signaling.

REFERENCES

  1. 1. Ferlay J, Parkin DM, Steliarova-Foucher E. Estimates of cancer incidence and mortality in Europe in 2008. Eur J Cancer. 2010; 46(4): 765781, doi: 10.1016/j.ejca.2009.12.014, indexed in Pubmed: 20116997.
  2. 2. Vaughan S, Coward JI, Bast RC, et al. Rethinking ovarian cancer: recommendations for improving outcomes. Nat Rev Cancer. 2011; 11(10): 719725, doi: 10.1038/nrc3144, indexed in Pubmed: 21941283.
  3. 3. Vargas-Hernández VM, Moreno-Eutimio MA, Acosta-Altamirano G, et al. Management of recurrent epithelial ovarian cancer. Gland Surg. 2014; 3(3): 198202, doi: 10.3978/j.issn.2227-684X.2013.10.01, indexed in Pubmed: 25207212.
  4. 4. Fung-Kee-Fung M, Oliver T, Elit L, et al. Optimal chemotherapy treatment for women with recurrent ovarian cancer. Curr Oncol. 2007; 14(5): 195208, indexed in Pubmed: 17938703.
  5. 5. Raja FA, Chopra N, Ledermann JA, et al. Targeted trials in ovarian cancer. Gynecol Oncol. 2010; 119(1): 151156, doi: 10.1016/j.ygyno.2010.05.008, indexed in Pubmed: 20591473.
  6. 6. Takeyama H, Wakamiya N, O’Hara C, et al. Tumor necrosis factor expression by human ovarian carcinoma in vivo. Cancer Res. 1991; 51(16): 44764480, indexed in Pubmed: 1868469.
  7. 7. Ihnatko R, Kubes M. TNF signaling: early events and phosphorylation. Gen Physiol Biophys. 2007; 26(3): 159167, indexed in Pubmed: 18063842.
  8. 8. MacEwan DJ. TNF receptor subtype signalling: differences and cellular consequences. Cell Signal. 2002; 14(6): 477492, indexed in Pubmed: 11897488.
  9. 9. Chen G, Goeddel DV. TNF-R1 signaling: a beautiful pathway. Science. 2002; 296(5573): 16341635, doi: 10.1126/science.1071924, indexed in Pubmed: 12040173.
  10. 10. Kazimierczuk Z, Górska A, Switaj T, et al. Adamantylaminopyrimidines and -pyridines are potent inducers of tumor necrosis factor-alpha. Bioorg Med Chem Lett. 2001; 11(9): 11971200, indexed in Pubmed: 11354376.
  11. 11. Mauri JK, Lasek W, Górska A, et al. Synthesis, structure and tumour necrosis factor-alpha production-enhancing properties of novel adamantylamino heterocyclic derivatives. Anticancer Drug Des. 2001; 16(2-3): 7380, indexed in Pubmed: 11962515.
  12. 12. Lasek W, Switaj T, Sieńko J, et al. Stimulation of TNF-alpha production by 2-(1-adamantylamino)-6-methylpyridine (AdAMP) - a novel immunomodulator with potential application in tumour immunotherapy. Cancer Chemother Pharmacol. 2002; 50(3): 213222, doi: 10.1007/s00280-002-0496-5, indexed in Pubmed: 12203103.
  13. 13. Ferreira JJ, Rascol O. Prevention and therapeutic strategies for levodopa-induced dyskinesias in Parkinson’s disease. Curr Opin Neurol. 2000; 13(4): 431436, indexed in Pubmed: 10970061.
  14. 14. Khare MD, Sharland M. Influenza. Expert Opin Pharmacother. 2000; 1(3): 367375, doi: 10.1517/14656566.1.3.367, indexed in Pubmed: 11249523.
  15. 15. Basarić N, Sohora M, Cindro N, et al. Antiproliferative and antiviral activity of three libraries of adamantane derivatives. Arch Pharm (Wein- heim). 2014; 347(5): 334340, doi: 10.1002/ardp.201300345, indexed in Pubmed: 24532384.
  16. 16. Aggarwal S, Takada Y, Singh S, et al. Activation of transcription factor NF-kappa B is suppressed by curcumin (diferuloylmethane) [corrected]. J Biol Chem. 1995; 270(42): 2499525000, indexed in Pubmed: 7559628.
  17. 17. Liu SF, Ye X, Malik AB. Inhibition of NF-kappaB activation by pyrrolidine dithiocarbamate prevents In vivo expression of proinflammatory genes. Circulation. 1999; 100(12): 13301337, indexed in Pubmed: 10491379.
  18. 18. Gilmore TD, Koedood M, Piffat KA, et al. Rel/NF-kappaB/IkappaB proteins and cancer. Oncogene. 1996; 13(7): 13671378, indexed in Pubmed: 8875974.
  19. 19. Shames BD, Selzman CH, Meldrum DR, et al. Interleukin-10 stabilizes inhibitory kappaB-alpha in human monocytes. Shock. 1998; 10(6): 389394, indexed in Pubmed: 9872676.
  20. 20. Voboril R, Weberova-Voborilova J. Sensitization of colorectal cancer cells to irradiation by IL-4 and IL-10 is associated with inhibition of NF-kappaB. Neoplasma. 2007; 54(6): 495502, indexed in Pubmed: 17949233.
  21. 21. Mitsiades N, Mitsiades CS, Richardson PG, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood. 2003; 101(6): 23772380, doi: 10.1182/blood-2002-06-1768, indexed in Pubmed: 12424198.
  22. 22. Kazimierczuk Z, Górska A, Switaj T, et al. Adamantylaminopyrimidines and -pyridines are potent inducers of tumor necrosis factor-alpha. Bioorg Med Chem Lett. 2001; 11(9): 11971200, indexed in Pubmed: 11354376.
  23. 23. Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res. 2010; 70(2): 440446, doi: 10.1158/0008-5472.CAN-09-1947, indexed in Pubmed: 20068163.
  24. 24. Knutson KL, Karyampudi L, Lamichhane P, et al. Targeted immune therapy of ovarian cancer. Cancer Metastasis Rev. 2015; 34(1): 5374, doi: 10.1007/s10555-014-9540-2, indexed in Pubmed: 25544369.
  25. 25. Pazhang Y, Jaliani HZ, Imani M, et al. Synergism between NF-kappa B inhibitor, celastrol, and XIAP inhibitor, embelin, in an acute myeloid leukemia cell line, HL-60. J Cancer Res Ther. 2016; 12(1): 155160, doi: 10.4103/0973-1482.150407, indexed in Pubmed: 27072230.
  26. 26. Scallon BJ, Moore MA, Trinh H, et al. Chimeric anti-TNF-alpha monoclonal antibody cA2 binds recombinant transmembrane TNF-alpha and activates immune effector functions. Cytokine. 1995; 7(3): 251259, doi: 10.1006/cyto.1995.0029, indexed in Pubmed: 7640345.
  27. 27. Lügering A, Schmidt M, Lügering N, et al. Infliximab induces apoptosis in monocytes from patients with chronic active Crohn’s disease by using a caspase-dependent pathway. Gastroenterology. 2001; 121(5): 11451157, indexed in Pubmed: 11677207.
  28. 28. Di Sabatino A, Pender SLF, Jackson CL, et al. Functional modulation of Crohn’s disease myofibroblasts by anti-tumor necrosis factor antibodies. Gastroenterology. 2007; 133(1): 137149, doi: 10.1053/j.gastro.2007.04.069, indexed in Pubmed: 17631138.
  29. 29. Rath PC, Aggarwal BB. TNF-induced signaling in apoptosis. J Clin Immunol. 1999; 19(6): 350364, indexed in Pubmed: 10634209.

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