Esra Münire CÜCE AYDOĞMUŞ, Gunseli Ayse INHAN GARIP

MCF 7 Hücre Hattında 4- Aminopiridin ve Paklitakselin Sinerjistik Etkileri

Amaç: Çalışmamızda, meme kanseri tedavisinde yaygın olarak kullanılan Paklitaksel’in (PTX) etkinliğinin 4-aminopiridin (4-AP) kullanılarak arttırılması hedeflenmiştir. Yöntemler: Çalışmamızda, L929 (ATCC CRL-6364) ve MCF7 (ATCC- HTB 22) hücre hatları kullanıldı. IC50 ve canlılık tripan mavisi metoduyla, hücre döngüsü analizleri Cdk2 ve Histon (H3) seviyeleri ile ve membran potansiyeli ölçümleri Dibac4(3) [Bis-(1,3-dibutylbarbituric acid)trimethine oxonol] kullanılarak floresan mikroplaka okuyucuda belirlendi.Bulgular: Yapılan deneylerde, her iki ajan için IC50 değerleri belirlendi ve bu değerler kombine edildi. Kombinasyonlarda iki farklı PTX konsantrasyonu kullanıldı. Kombinasyon sonucunda, L929 hücrelerinde 4-AP (4 mM) ve PTX (5 nM), 4-AP (4 mM) ve PTX (7.5 nM) kombinasyonunun canlılığı sırasıyla % 17 ± 8.08, % 45 ± 3.18, MCF 7 hücrelerinde ise sırasıyla %60 ± 3.7, %74 ± 2.6 azalttığı belirlenmiştir. Membran potansiyeli (Vm) ölçümlerinde, L929 hücreleri için 4-AP ve 5 nM- 7.5 nM PTX değerlerinde depolarizasyon, kombinasyonlar için hiperpolarizasyon gözlemlenirken, MCF7 hücrelerinde 4-AP,7.5nM PTX ve 4-AP (4 mM) + PTX (5 nM) için depolarizasyon, 5 nM PTX ve 4-AP (4 mM) + PTX (7.5 nM) için hyperpolarizasyon gözlemlenmiştir. Cdk2 ve H3 seviyeleri ölçümlerinde, MCF 7 hücreleri için ağırlıklı olarak G1 fazında, L929 hücreleri ağırlıklı olarak G2/ M fazında duraklama görülmüştür. Sonuç: MCF-7 meme kanseri hücrelerinde 4-AP ve PTX in beraber kullanılmasıyla canlılıkta azalma beklendiği şekilde artarken, sağlıklı hücre hattı olarak kullanılan L929 hücrelerinde antagonist etki görülmüştür. Bu veriler, 4- AP ile PTX kombinasyonunun kanser hücrelerini seçici olarak etkilediğini ve dolayısıyla umut vadeden bir yaklaşım olduğunu göstermektedir.

Anahtar Kelimeler:

4- aminopiridine, Paklitaksel, MCF7 meme kanseri hücre hattı

Synergistic Effects of 4-Aminopyridine and Paclitaxel on MCF 7 Cell Line

Objectives: Aim of this study is to increase the effectiveness of paclitaxel (PTX) with the use of 4 – aminopyridine (4-AP) on breast cancer cell line MCF-7.Methods: In this study, L-929 (ATCC CRL-6364) and MCF-7 (ATCC – HTB 22) cell lines were used. IC50 and survival values were determined by trypan blue exclusion; cell cycle analysis was determined by measuring levels of Cdk2 and Histone (H3) and plasma membrane potential (Vm) measurements were performed using fluorescent Bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBaC4(3)).Results: IC50 values were determined for two agents and these values were combined. Combination treatments ie. “4-AP (4 mM) + PTX (5 nM)” and “4-AP (4 mM) + PTX (7.5 nM)” decreased viability 17% ± 8.08 and 45% ± 3.18, respectively for L-929 cells and decreased viability 60%± 3.7 and 74%± 2.6, respectively for MCF-7 cells. For L-929 cells, plasma membrane potential measurements resulted in depolarization for 4-AP, PTX (5 nM) and PTX (7.5 nM), and resulted in hyperpolarization for the combinations. For MCF-7 cells, plasma membrane potential measurements resulted in depolarization for 4-AP, PTX (7.5 nM) and 4-AP + PTX (5 nM), and resulted in hyperpolarization for PTX (5 nM) and 4-AP + PTX (7.5 nM). Changes of Cdk2 and H3 levels showed mostly G1 arrest for MCF 7 cells and G2/M arrest for L-929 cells.Conclusions: Combination treatments increased the cell death for MCF-7 cells. But, combination treatments didn’t show synergistic effect on L-929 which is accepted as a non-cancerous cell. These data showed that use of 4-AP in combination with the anticancer agent paclitaxel is a promising approach for cancer treatment.

Keywords:

4- aminopyridine, Paclitaxel, MCF 7 breast cancer cell line,

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1.Sharma G. N., Dave R, Sanadya J, Sharma P, Sharma K. K. Various Types and Management of Breast Cancer: an Overview. J Adv Pharm Technol Res 2010; 1(2): 109–126.
2.Adrienne G. Waks, MD; Eric P. Winer. Breast Cancer Treatment A Review. JAMA 2019; 321(3):288-300.
3. Walsh V, Goodman J. The billion dollar molecule: Taxol in historical and theoretical perspective. Clio Med 2002; 66: 245–267.
4. Erdemoğlu N, Şener B. Taksan Sınıfı Bileşiklerin Antitümör Etkileri. Ankara Ecz. Fak. Derg 2000; 29(1): 77-90.
5. Jordan M. A., Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer2004; 4(4): 253–265.
6. Weaver BA. How Taxol/paclitaxel kills cancer cells. Mol Biol Cell 2014;25(18):2677–2681.
7. Bates E. Ion Channels in Development and Cancer. Annual Review of Cell and Developmental Biology 2015; 31(1): 231-247.
8. Comes N., Serrano-Albarrás A., Capera J., Serrano-Novillo C., Condom E., Ramón y Cajal S., Ferreres J.C., Felipe A. Involvement of potassium channels in the progression of cancer to a more malignant phenotype. Biochim. Biophys. Acta Biomembr. 2015; 1848: 2477–2492.
9. Jensen JM, Shi R. Effects of 4-aminopyridine on stretched mammalian spinal cord: the role of potassium channels in axonal conduction. J Neurophysiol 2003 Oct; 90(4): 2334-40.
10. Wang W, Fan Y, Wang S, et al. Effects of voltage-gated K+ channel on cell proliferation in multiple myeloma. ScientificWorldJournal. 2014;2014:785140. doi:10.1155/2014/785140
11. Kim JA, Kang YS, Jung MW, Kang GH, Lee SH, Lee YS. Ca2+ influx mediates apoptosis induced by 4-aminopyridine, a K+ channel blocker, in HepG2 human hepatoblastoma cells. Pharmacology 2000 Feb; 60(2): 74-81.
12. Wang W, Xiao j, Adachi M, Liu Z, Zhou J. 4-aminopyridine Induces Apoptosis of Human Acute Myeloid Leukemia Cells via Increasing [Ca2+] i Through P2 X7 Receptor Pathway. Cell Physiol Biochem 2011; 28: 199-208.
13. Hu C.L., Liu Z., Zeng X.M., Liu Z.Q., Chen X.H., Zhang Z.H., Mei Y.A. 4-Aminopyridine, a Kv channel antagonist, prevents apoptosis of rat cerebellar granule neurons. Neuropharmacology 2006; 51: 737–746.
14. Barghouth PG, Thiruvalluvan M, Oviedo NJ. Bioelectrical regulation of cell cycle and the planarian model system. Biochimica et Biophysica Acta 2015;1848: 2629–2637.
15. Sundelacruz S, Levin M, Kaplan DL. Role of Membrane Potential in the Regulation of Cell Proliferation and Differentiation. Stem Cell Rev 2009 Sep; 5(3): 231-46.
16. Yusuf R.Z., Duan Z., Lamendola D.E., Penson R.T. and Seiden M.V. Paclitaxel Resistance: Molecular Mechanisms and Pharmacologic Manipulation. Current Cancer Drug Targets 2003; 3: 1-19.
17.Yardley D. A. Drug Resistance and the Role of Combination Chemotherapy in Improving Patient Outcomes. International Journal of Breast Cancer 2013. doi:org/10.1155/2013/137414.
18. Huang X, Jan LY. Targeting potassium channels in cancer. J Cell Biol. 2014;206(2):151–162. doi:10.1083/jcb.201404136
19. Aromolaran K.A., Goldstein PA. Ion channels and neuronal hyperexcitability in chemotherapy-induced peripheral neuropathy: Cause and effect? Molecular Pain 2017;13:1–24.