Investigation Of The Anti-Proliferative and Anti-Apoptotic Effects Of Boric Acid On Human Non-Small Cell Lung Cancer Cells Through The TGF-β Signaling Pathway

Öz

Objective: Lung cancer is the most common type of cancer in both men and women worldwide. Despite scientific advances in cancer in recent years, this pathology still ranks first with high mortality rates. Non-small cell lung cancer constitutes 85% of lung cancer cases. In these patients, the course of the disease progresses very poorly and since the diagnosis is mostly made in advanced stages, the treatments applied in most patients are not successful. Boric acid (BA) is an essential microelement with its chemical properties, low side effects and anti-carcinogenic effects. In this study, it was aimed to investigate the cytotoxic, anti-proliferative and apoptotic effects of Boric Acid (BA), which has known anti-proliferative and anti-apoptotic effects, on non-small cell lung cancer cells (A549) via TGF-β signaling pathway. Methods: Human non-small cell lung cancer cells (A549) were used in the study. Cytotoxic analysis of BA was performed by MTT analysis. The apoptotic effect of BA was determined by Annexin V/PI and immunofluorescence analysis. Expression levels of TGF-β, SMAD2/3/4 genes were analyzed at the molecular level by quantitative real-time polymerase chain reaction (RT-qPCR). Each reaction was replicated three times. Results: As a result of MTT analysis, it was observed that 50 mM BA decreased proliferation in A549 cells (p<0.01). With 50 mM BA application, the rate of apoptotic cells at 48 hours increased to 9.7%, and the rate of cells in the early apoptosis stage to 10.1%. In the immunofluorescence analysis, it was observed that the level of Caspase-3 labeling index in A549 cells was higher than the control group. No significant change was observed in the expression of SMAD3 and SMAD4 genes in BA applied A549 cells. While a decrease was observed in the expression of TGF-β gene (p<0.05), an increase in SMAD2 gene expression was observed (p<0.01). Conclusion: The results show that BA may be a new therapeutic agent with anti-proliferative and anti-apoptotic activity. However, further in vivo and advanced molecular level studies are needed to support these findings.

Anahtar Kelimeler

• Non-small cell lung cancer
• boric acid
• cytotoxicity
• apoptosis
• TGF-β/Smad signaling pathway

Borik Asitin Anti-Proliferatif ve Anti-Apoptotik Etkilerinin İnsan Küçük Hücreli Dışı Akciğer Kanseri Hücrelerinde TGF-β Sinyal Yolağı Üzerinden İncelenmesi

Öz

Amaç: Akciğer kanseri, tüm dünyada hem kadın hem de erkeklerde en sık görülen kanser türüdür. Son yıllarda kanser konusunda elde edilen bilimsel gelişmelere rağmen, bu patoloji hala yüksek mortalite oranları ile ilk sıralarda yer almaktadır. Akciğer kanseri olgularının %85’ini küçük hücreli olmayan akciğer kanseri oluşturmaktadır. Bu hastalarda hastalığın seyri oldukça kötü ilerlemekte ve tanısı çoğunlukla ileri evrelerde konulduğundan, çoğu hastada uygulanan tedaviler başarılı olamamaktadır. Borik asit (BA) sahip olduğu kimyasal özellikler, düşük yan etki oranları ve anti-kanserojen etkilere sahip esansiyel bir mikro elementtir. Bu çalışma kapsamında anti-proliferatif ve anti-apoptotik etkileri bilinen BA’nın, küçük hücreli dışı akciğer kanseri hücreleri (A549) üzerindeki sitotoksik, anti-proliferatif ve apoptotik etkilerinin TGF-β sinyal yolağı üzerinden araştırılması amaçlanmıştır. Yöntem: Çalışmada insan küçük hücreli dışı akciğer kanseri hücreleri (A549) kullanıldı. BA’nın sitotoksik analizi MTT analizi ile yapıldı. BA’nın apoptotik etkisi Annexin V/PI ve immünfloresan analizlerle belirlendi. TGF-β, SMAD2/3/4 genlerinin ekspresyon düzeyleri moleküler düzeyde kantitatif gerçek zamanlı polimeraz zincir reaksiyonu (RT-qPCR) ile analiz edildi. Her bir reaksiyon üç kez tekrarlandı. Bulgular: MTT analizi sonucu 50 mM BA’nın A549 hücrelerinde proliferasyonu azalttığı gözlendi (p<0.01). 50 mM BA uygulaması ile 48. saatte apoptotik hücre oranı %9.7’ye, erken apoptoz evresindeki hücre oranı ise %10.1’e yükseldi. İmmünfloresan analizinde de A549 hücrelerinde Kaspaz-3 işaretlenme düzeyininin kontrol grubuna göre yüksek miktarda olduğu gözlendi. BA uygulanan A549 hücrelerinde SMAD3 ve SMAD4 genlerinin ifadesinde anlamlı bir değişme gözlenmedi. TGF-β geninin ifadesinde azalma gözlenirken (p<0.05), SMAD2 gen ifadesinde artış gözlendi (p<0.01). Sonuç: Sonuçlar BA’nın; anti-proliferatif ve anti-apoptotik aktiviteye sahip yeni bir tedavi edici ajan olabileceğini göstermektedir. Ancak bu etkilere aracılık eden yolakların aydınlatılması için ileri düzeyde çalışmalara ihtiyaç bulunmaktadır.

Anahtar Kelimeler

• Küçük hücreli dışı akciğer kanseri
• borik asit
• sitotoksisite
• apoptoz
• TGF-β/Smad sinyal yolağı

Kaynakça

1. 1. Bade, B. C., & Dela Cruz, C. S. (2020). Lung Cancer 2020: Epidemiology, Etiology, and Prevention. Clin Chest Med, 41(1), 1-24. doi:10.1016/j.ccm.2019.10.001.
2. 2. Nasim, F., Sabath, B. F., & Eapen, G. A. (2019). Lung Cancer. Med Clin North Am, 103(3), 463-473. doi:10.1016/j.mcna.2018.12.006
3. 3. HerbstRS, M. (2018). BoshoffC. The Biology and Management of Non-Small Cell Lung Cancer. Nature, 553(7689), 446-454.
4. 4. Alexander, M., Kim, S. Y., & Cheng, H. (2020). Update 2020: management of non-small cell lung cancer. Lung, 198, 897-907.
5. 5. Molina, N. D. R. S. D. (2019). JR. Non-Small Cell Lung Cancer: Epidemiology, Screening, Diagnosis, and Treatment [Internet]. Mayo Clin. Proc. Elsevier Ltd, 1623-1640.
6. 6. Pirker, R. (2020). Chemotherapy remains a cornerstone in the treatment of nonsmall cell lung cancer. Current opinion in oncology, 32(1), 63-67.
7. 7. Lv, P., Man, S., Xie, L., Ma, L., & Gao, W. (2021). Pathogenesis and therapeutic strategy in platinum resistance lung cancer. Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, 1876(1), 188577.
8. 8. El-Hussein, A., Manoto, S. L., Ombinda-Lemboumba, S., Alrowaili, Z. A., & Mthunzi-Kufa, P. (2021). A review of chemotherapy and photodynamic therapy for lung cancer treatment. Anti-Cancer Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Anti-Cancer Agents), 21(2), 149-161.

9. 9. Sjøgren, K., Jacobsen, K. A., Grønberg, B. H., & Halvorsen, T. O. (2020). Timing of severe toxicity from chemotherapy in patients with lung cancer. Anticancer research, 40(11), 6399-6406.
10. 10. Hirsch, F., Scagliotti, G., Mulshine, J., & Kwon, R., Curran W.J., Wu YL, et al. (2017). Lung cancer: current therapies and new targeted treatments. Lancet, 389(10066), 299-311.
11. 11. Corti, A., Dominici, S., Piaggi, S., & Pompella, A. (2023). Enhancement of ferroptosis by boric acid and its potential use as chemosensitizer in anticancer chemotherapy. BioFactors, 49(2), 405-414.
12. 12. Mahabir, S., Spitz, M., Barrera, S., Dong, Y., Eastham, C., & Forman, M. (2008). Dietary boron and hormone replacement therapy as risk factors for lung cancer in women. American journal of epidemiology, 167(9), 1070-1080.
13. 13. Korkmaz, M., Uzgören, E., Bakırdere, S., Aydın, F., & Ataman, O. Y. (2007). Effects of dietary boron on cervical cytopathology and on micronucleus frequency in exfoliated buccal cells. Environmental toxicology, 22(1), 17-25.
14. 14. Cui, Y., Winton, M. I., Zhang, Z.-F., Rainey, C., Marshall, J., De Kernion, J. B., et al. (2004). Dietary boron intake and prostate cancer risk. Oncology reports, 11(4), 887-892.
15. 15. Barranco, W. T., & Eckhert, C. D. (2004). Boric acid inhibits human prostate cancer cell proliferation. Cancer letters, 216(1), 21-29.
16. 16. Kahraman, E., & Göker, E. (2022). Boric acid exert anti-cancer effect in poorly differentiated hepatocellular carcinoma cells via inhibition of AKT signaling pathway. Journal of Trace Elements in Medicine and Biology, 73, 127043.
17. 17. Sevimli, M., Bayram, D., Özgöçmen, M., Armağan, I., & Semerci Sevimli, T. (2022). Boric acid suppresses cell proliferation by TNF signaling pathway mediated apoptosis in SW-480 human colon cancer line. J Trace Elem Med Biol, 71, 126958. doi:10.1016/j.jtemb.2022.126958.
18. 18. Cebeci, E., Yüksel, B., & Şahin, F. (2022). Anti-cancer effect of boron derivatives on small-cell lung cancer. Journal of Trace Elements in Medicine and Biology, 70, 126923.
19. 19. Scorei, R., Ciubar, R., Ciofrangeanu, C. M., Mitran, V., Cimpean, A., & Iordachescu, D. (2008). Comparative effects of boric acid and calcium fructoborate on breast cancer cells. Biological trace element research, 122(3), 197-205.
20. 20. Acerbo, A. S., & Miller, L. M. (2009). Assessment of the chemical changes induced in human melanoma cells by boric acid treatment using infrared imaging. Analyst, 134(8), 1669-1674.
21. 21. Aydin, H. E., Gunduz, M. K., Kizmazoglu, C., Kandemir, T., & Arslantas, A. (2021). Cytotoxic effect of boron application on glioblastoma cells. Turk Neurosurg, 31(2), 206-210.
22. 22. Massagué, J. (2012). TGFβ signalling in context. Nature reviews Molecular cell biology, 13(10), 616-630.
23. 23. Soriano-Ursúa, M. A., Farfán-García, E. D., & Geninatti-Crich, S. (2019). Turning fear of boron toxicity into boron-containing drug design. Current medicinal chemistry, 26(26), 5005-5018.
24. 24. Dymova, M. A., Taskaev, S. Y., Richter, V. A., & Kuligina, E. V. (2020). Boron neutron capture therapy: Current status and future perspectives. Cancer communications, 40(9), 406-421.
25. 25. Miao, S., Ge, Y., Yi, Z., & Feng, Q. (2020). Screening of Aptamer for Breast Cancer Biomarker Calreticulin and Its Application to Detection of Serum and Recognition of Breast Cancer Cell. Chinese Journal of Analytical Chemistry, 48(5), 642-649.
26. 26. Simsek, F., Inan, S., & Korkmaz, M. (2019). An in vitro study in which new boron derivatives maybe an option for breast cancer treatment. Breast cancer, 13, 14.
27. 27. Söğüt, Ö., & Acar, O. (2020). Bor ve sağlık. Literatür Eczacılık Bilimleri Dergisi, 9(1), 11-17.
28. 28. Turkez, H., Tatar, A., Hacımuftuoglu, A., & Ozdemir, E. (2010). Boric acid as a protector against paclitaxel genotoxicity. Acta Biochimica Polonica, 57(1), 95-97.
29. 29. Hazman, Ö., Bozkurt, M. F., Fidan, A. F., Uysal, F. E., & Çelik, S. (2018). The effect of boric acid and borax on oxidative stress, inflammation, ER stress and apoptosis in cisplatin toxication and nephrotoxicity developing as a result of toxication. Inflammation, 41, 1032-1048.
30. 30. Fırat, F., & Aladağ, T. (2022). Comparatıve effects of borıc acıd and resveratrol on mcf-7 breast cancer cells metastatıc behaviour. International Journal of Research - GRANTHAALAYAH, 10(1), 34-46. doi: 10.29121/granthaalayah.v10.i1.2022.4460
31. 31. Ersöz, M., Kaçar, A. K., Sezekler, I., & Coşkun, Z. M. (2019). Effects of boric-acid-applied green tea on the expressions of heat shock proteins in MCF-7 cells. Cukurova Medical Journal, 44(1), 66-71.
32. 32. Gallardo-Williams, M. T., Chapin, R. E., King, P. E., Moser, G. J., Goldsworthy, T. L., Morrison, J. P., et al. (2004). Boron supplementation inhibits the growth and local expression of IGF-1 in human prostate adenocarcinoma (LNCaP) tumors in nude mice. Toxicologic pathology, 32(1), 73-78.
33. 33. Zafar, H., & Ali, S. (2013). Boron inhibits the proliferating cell nuclear antigen index, molybdenum containing proteins and ameliorates oxidative stress in hepatocellular carcinoma. Archives of biochemistry and biophysics, 529(2), 66-74.
34. 34. Liu, L.C., Tsao, T. C.-Y., Hsu, S.-R., Wang, H.-C., Tsai, T.-C., Kao, J.-Y., et al. (2012). EGCG inhibits transforming growth factor-β-mediated epithelial-to-mesenchymal transition via the inhibition of Smad2 and Erk1/2 signaling pathways in nonsmall cell lung cancer cells. Journal of Agricultural and Food Chemistry, 60(39), 9863-9873.
35. 35. Li, Q., Zhang, D., Wang, Y., Sun, P., Hou, X., Larner, J., et al. (2013). MiR-21/Smad 7 signaling determines TGF-β1-induced CAF formation. Scientific reports, 3(1), 2038.
36. 36. Tirino, V., Camerlingo, R., Bifulco, K., Irollo, E., Montella, R., Paino, F., . et al.(2013). TGF-β1 exposure induces epithelial to mesenchymal transition both in CSCs and non-CSCs of the A549 cell line, leading to an increase of migration ability in the CD133+ A549 cell fraction. Cell death & disease, 4(5), e620-e620.
37. 37. Derynck, R., Turley, S. J., & Akhurst, R. J. (2021). TGFβ biology in cancer progression and immunotherapy. Nature Reviews Clinical Oncology, 18(1), 9-34.
38. 38. Wiercinska, E., Naber, H. P., Pardali, E., van der Pluijm, G., van Dam, H., & Ten Dijke, P. (2011). The TGF-β/Smad pathway induces breast cancer cell invasion through the up-regulation of matrix metalloproteinase 2 and 9 in a spheroid invasion model system. Breast cancer research and treatment, 128, 657-666.
39. 39. Kretzschmar, M. (2000). Transforming growth factor-β and breast cancer: transforming growth factor-β/SMAD signaling defects and cancer. Breast Cancer Research, 2(2), 1-9.
40. 40. Zheng, L., Liang, H., Zhang, Q., Shen, Z., Sun, Y., Zhao, X., .et al.(2022). circPTEN1, a circular RNA generated from PTEN, suppresses cancer progression through inhibition of TGF-β/Smad signaling. Molecular cancer, 21(1), 41.
41. 41. Peng, Y., Fu, Z.-z., Guo, C.-S., Zhang, Y.-X., Di, Y., Jiang, B., et al.(2015). Effects and mechanism of baicalin on apoptosis of cervical cancer HeLa cells in-vitro. Iranian journal of pharmaceutical research: IJPR, 14(1), 251.
42. 42. Güleş, Ö., & Ülker, E. (2008). Apoptozun belirlenmesinde kullanılan yöntemler. Yüzüncü Yıl Üniversitesi Veteriner Fakültesi Dergisi, 19(2), 73-78.