Advances in Molecular OncologyAdvances in Molecular OncologyУспехи молекулярной онкологии2313-805X2413-3787Publishing House ABV Press10410.17650/2313-805X-2017-4-3-75-82RESEARCH ARTICLESЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИThe role of autophagy inhibition in the enhanced cytotoxicity of temozolomide on melanoma cell linesРоль ингибирования аутофагии в изменении цитотоксичности темозоломида на клеточных линиях меланомыRyabayaO. O.РябаяО. О.
Russian Federation

1 Ostrovityanovа St., Moscow 117997.

117997 Москва, ул. Островитянова, 1.

oxa2601@yandex.ruInshakovA. N.ИншаковА. Н.
Russian Federation

24 Kashirskoye Shosse, Moscow 115478.

115478 Москва, Каширское шоссе, 24.

MalyshevaA. A.МалышеваА. А.
Russian Federation

24 Kashirskoye Shosse, Moscow 115478.

115478 Москва, Каширское шоссе, 24.

AbramovI. S.АбрамовИ. С.
Russian Federation

32 Vavilova St., Moscow 119991.

119991 Москва, ул. Вавилова, 32.

SholinaN. V.ШолинаН. В.
Russian Federation

24 Kashirskoye Shosse, Moscow 115478.

115478 Москва, Каширское шоссе, 24.

KhochenkovD. A.ХоченковД. А.
Russian Federation

24 Kashirskoye Shosse, Moscow 115478.

115478 Москва, Каширское шоссе, 24.

StepanovaE. V.СтепановаЕ. В.
Russian Federation

24 Kashirskoye Shosse, Moscow 115478.

115478 Москва, Каширское шоссе, 24.

N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of RussiaФГБУ «Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Минздрава РоссииN.I. Pirogov Russian National Research Medical University, Ministry of Health of Russia.ФГБОУ ВО «Российский национальный исследовательский медицинский университет им. Н.И. Пирогова» Минздрава России.N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia.ФГБУ «Национальный медицинский исследовательский центр онкологии им. Н.Н. Блохина» Минздрава России.V.A. Engelhardt Institute of Molecular Biology of the Russian Academy of Sciences.ФГБУН «Институт молекулярной биологии им. В.А. Энгельгардта РАН».150920174375821610201716102017Copyright ©; 2017, Ryabaya O.O., Inshakov A.N., Malysheva A.A., Abramov I.S., Sholina N.V., Khochenkov D.A., Stepanova E.V.Copyright ©; 2017, Рябая О.О., Иншаков А.Н., Малышева А.А., Абрамов И.С., Шолина Н.В., Хоченков Д.А., Степанова Е.В.2017Ryabaya O.O., Inshakov A.N., Malysheva A.A., Abramov I.S., Sholina N.V., Khochenkov D.A., Stepanova E.V.Рябая О.О., Иншаков А.Н., Малышева А.А., Абрамов И.С., Шолина Н.В., Хоченков Д.А., Степанова Е.В.https://creativecommons.org/licenses/by/4.0https://umo.abvpress.ru/jour/article/view/104

Background. Despite advantages in treatment of metastatic melanoma it remains resistant to current therapy. Recent evidence indicates that tumor cells could overcome death through autophagy, a process that degrades cellular proteins and organelles to maintain cellular biosynthesis during nutrient deprivation or lack of energy. Objective: to investigate the involvement of autophagy inhibitors chloroquine (CQ) and LY-294.002 (LY) in temozolomide (TMZ) cytotoxicity in human melanoma cell lines.

Materials and methods. The study was performed on patient-derived melanoma cell lines Mel Z, Mel IL and Mel MTP. The antiproliferative activity of combined TMZ and autophagy inhibitors treatment was determined by MTT assay and colony-forming assay. Cell cycle analysis, apoptosis activation and expression analysis of key autophagy markers under combined treatment was evaluated.

Results. CQ and LY enhanced the cytotoxicity of TMZ and reduced colony formation in 3 melanoma cell lines, moreover both inhibitors increased cell population in G0 / G1 phase of cell cycle in Mel Z, Mel IL cell lines, but not in Mel MTP. CQ and LY synergistically activated apoptosis in all cell lines. The matrix RNA expression analysis of key autophagy genes showed autophagy involvement in enhanced cytotoxicity.

Conclusions. Thus, autophagy inhibition on different stages of this process could overcome resistance to TMZ and be applicable as potent target in metastatic melanoma treatment.

melanomaautophagyapoptosischemoresistancetemozolomidechloroquineLY-294.002меланомааутофагияапоптозхимиорезистентностьтемозоломидхлорокинLY-294.0021. Saito R.F., Tortelli T.C. Jr, Jacomassi M.D. et al. Emerging targets for combination therapy in melanomas. FEBS Lett 2015;589(22):3438–48.Saito R.F., Tortelli T.C. Jr, Jacomassi M.D. et al. Emerging targets for combination therapy in melanomas. FEBS Lett 2015;589(22):3438–48.2. Soura E., Eliades P.J., Shannon K. et al. Hereditary melanoma: update on syndromes and management: emerging melanoma cancer complexes and genetic counseling. J Am Acad Dermatol 2016;74(3):411–20.Soura E., Eliades P.J., Shannon K. et al. Hereditary melanoma: update on syndromes and management: emerging melanoma cancer complexes and genetic counseling. J Am Acad Dermatol 2016;74(3):411–20.3. Luke J., Schwartz G. Chemotherapy in the management of advanced cutaneous malignant melanoma. Clin in Dermat 2013;31(3):290–7.Luke J., Schwartz G. Chemotherapy in the management of advanced cutaneous malignant melanoma. Clin in Dermat 2013;31(3):290–7.4. Yeramian A., Sorolla A., Velasco A. et al. Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway. Int J Cancer 2012;130:967–78.Yeramian A., Sorolla A., Velasco A. et al. Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway. Int J Cancer 2012;130:967–78.5. Zhang J., Stevens M.F., Bradshaw T.D. Temozolomide: mechanisms of action, repair and resistance. Curr Mol Pharmacol 2012;5(1):102–14.Zhang J., Stevens M.F., Bradshaw T.D. Temozolomide: mechanisms of action, repair and resistance. Curr Mol Pharmacol 2012;5(1):102–14.6. Denny B.J., Wheelhouse R.T., Stevens M.F. et al. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry 1994;33(31):9045–51.Denny B.J., Wheelhouse R.T., Stevens M.F. et al. NMR and molecular modeling investigation of the mechanism of activation of the antitumor drug temozolomide and its interaction with DNA. Biochemistry 1994;33(31):9045–51.7. Hirose Y., Berger M.S., Pieper R.O. p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 2001;61(5):1957–63.Hirose Y., Berger M.S., Pieper R.O. p53 effects both the duration of G2/M arrest and the fate of temozolomide-treated human glioblastoma cells. Cancer Res 2001;61(5):1957–63.8. Kanzawa T., Germano I.M., Komata T. et al. Role of autophagy in temozolomideinduced cytotoxicity for malignant glioma cells. Cell Death Differ 2004;1(4): 448–57.Kanzawa T., Germano I.M., Komata T. et al. Role of autophagy in temozolomideinduced cytotoxicity for malignant glioma cells. Cell Death Differ 2004;1(4): 448–57.9. Rangwala R., Leone R., Chang Y.C. et al. Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma. Autophagy 2014;10(8):1369–79.Rangwala R., Leone R., Chang Y.C. et al. Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma. Autophagy 2014;10(8):1369–79.10. Mathew R., Karantza-Wadsworth V., White E. Role of autophagy in cancer. Nat Rev Cancer 2007;7(12):961–7.Mathew R., Karantza-Wadsworth V., White E. Role of autophagy in cancer. Nat Rev Cancer 2007;7(12):961–7.11. Maes H., Martin S., Verfaillie T., Agostinis P. Dynamic interplay between autophagic flux and Akt during melanoma progression in vitro. Exp Dermatol 2014;23(2):101–6.Maes H., Martin S., Verfaillie T., Agostinis P. Dynamic interplay between autophagic flux and Akt during melanoma progression in vitro. Exp Dermatol 2014;23(2):101–6.12. Lazova R., Camp R.L., Klump V. et al. Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome. Clin Cancer Res 2012;18(2):370–79.Lazova R., Camp R.L., Klump V. et al. Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome. Clin Cancer Res 2012;18(2):370–79.13. Guo J.Y., Chen H.Y., Mathew R. et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 2011;25(5):460–70.Guo J.Y., Chen H.Y., Mathew R. et al. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev 2011;25(5):460–70.14. Thorburn A., Morgan M.J. Targeting autophagy in BRAF mutant tumors. Cancer Discov 2015;5(4):353–4.Thorburn A., Morgan M.J. Targeting autophagy in BRAF mutant tumors. Cancer Discov 2015;5(4):353–4.15. National Library of Medicine US. Clinical trials investigating the use of chloroquine in cancer: NCT01575782, NCT00969306, NCT01446016, NCT01023477, NCT01469455, NCT01438177, NCT01727531, NCT00224978. Сlinicaltrials.gov: Bethesda, MD, 2013.National Library of Medicine US. Clinical trials investigating the use of chloroquine in cancer: NCT01575782, NCT00969306, NCT01446016, NCT01023477, NCT01469455, NCT01438177, NCT01727531, NCT00224978. Сlinicaltrials.gov: Bethesda, MD, 2013.16. National Library of Medicine US. Clinical trials investigating the use of hydroxychloroquine in cancer: NCT01273805, NCT00933803, NCT01006369, NCT01480154, NCT00765765, NCT00728845, NCT01266057, NCT00813423, NCT01494155, NCT01023737, NCT00786682, NCT00726596, NCT01417403, NCT00809237, NCT00909831, NCT00714181, NCT00714181, NCT01206530, NCT01026844, NCT00486603, NCT01649947, NCT00977470, NCT01506973, NCT01128296, NCT00568880, NCT01144169, NCT00962845, NCT01292408, NCT01689987, NCT01550367, NCT01396200, NCT01227135, NCT01602588, NCT01510119, NCT00031824, NCT01548768, NCT00908089, NCT01687179, NCT00405275, NCT00771056, NCT01709578. Сlinicaltrials.gov: Bethesda, MD, 2013.National Library of Medicine US. Clinical trials investigating the use of hydroxychloroquine in cancer: NCT01273805, NCT00933803, NCT01006369, NCT01480154, NCT00765765, NCT00728845, NCT01266057, NCT00813423, NCT01494155, NCT01023737, NCT00786682, NCT00726596, NCT01417403, NCT00809237, NCT00909831, NCT00714181, NCT00714181, NCT01206530, NCT01026844, NCT00486603, NCT01649947, NCT00977470, NCT01506973, NCT01128296, NCT00568880, NCT01144169, NCT00962845, NCT01292408, NCT01689987, NCT01550367, NCT01396200, NCT01227135, NCT01602588, NCT01510119, NCT00031824, NCT01548768, NCT00908089, NCT01687179, NCT00405275, NCT00771056, NCT01709578. Сlinicaltrials.gov: Bethesda, MD, 2013.17. Katayama M., Kawaguchi T., Berger M.S., Pieper R.O. DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ 2007;14(3):548–58.Katayama M., Kawaguchi T., Berger M.S., Pieper R.O. DNA damaging agent-induced autophagy produces a cytoprotective adenosine triphosphate surge in malignant glioma cells. Cell Death Differ 2007;14(3):548–58.18. Ma X.H., Piao S., Wang D. et al. Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma. Clin Cancer Res 2011;17(10):3478–89.Ma X.H., Piao S., Wang D. et al. Measurements of tumor cell autophagy predict invasiveness, resistance to chemotherapy, and survival in melanoma. Clin Cancer Res 2011;17(10):3478–89.19. Amaravadi R.K., Yu D., Lum J.J. et al., Autophagy inhibition enhances therapyinduced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 2007;117(2):326–36.Amaravadi R.K., Yu D., Lum J.J. et al., Autophagy inhibition enhances therapyinduced apoptosis in a Myc-induced model of lymphoma. J Clin Invest 2007;117(2):326–36.20. Guo X.L., Li D., Hu F. et al. Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells. Cancer Lett 2012;320(2):171–9.Guo X.L., Li D., Hu F. et al. Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells. Cancer Lett 2012;320(2):171–9.21. Carew J.S., Nawrocki S.T., Cleveland J.L. Modulating autophagy for therapeutic benefit. Autophagy 2007;3(5):464–7.Carew J.S., Nawrocki S.T., Cleveland J.L. Modulating autophagy for therapeutic benefit. Autophagy 2007;3(5):464–7.22. Lee S.W., Kim H.K., Lee N.H. et al. The synergistic effect of combination temozolomide and chloroquine treatment is dependent on autophagy formation and p53 status in glioma cells. Cancer Lett 2015;360(2):195–204.Lee S.W., Kim H.K., Lee N.H. et al. The synergistic effect of combination temozolomide and chloroquine treatment is dependent on autophagy formation and p53 status in glioma cells. Cancer Lett 2015;360(2):195–204.23. Carlino M.S., Todd J.R., Gowrishankar K. et al. Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma. Mol Oncol 2014;8(3):544–54.Carlino M.S., Todd J.R., Gowrishankar K. et al. Differential activity of MEK and ERK inhibitors in BRAF inhibitor resistant melanoma. Mol Oncol 2014;8(3):544–54.24. Yang Y.P., Hu L.F., Zheng H.F. et al. Application and interpretation of current autophagy inhibitors and activators. Acta Pharmacol Sin 2013;34(5): 625–35.Yang Y.P., Hu L.F., Zheng H.F. et al. Application and interpretation of current autophagy inhibitors and activators. Acta Pharmacol Sin 2013;34(5): 625–35.25. Davies H., Bignell G.R., Cox C. et al. Mutations of the BRAF gene in human cancer. Nature 2002;417(6892):949–54.Davies H., Bignell G.R., Cox C. et al. Mutations of the BRAF gene in human cancer. Nature 2002;417(6892):949–54.26. Armstrong J.L., Corazzari M., Martin S. et al. Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition. Clin Cancer Res 2011;17(8):2216–26.Armstrong J.L., Corazzari M., Martin S. et al. Oncogenic B-RAF signaling in melanoma impairs the therapeutic advantage of autophagy inhibition. Clin Cancer Res 2011;17(8):2216–26.27. Ma X.H., Piao S.F., Dey S. et al. Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma. J Clin Invest 2014;124(3):1406–17.Ma X.H., Piao S.F., Dey S. et al. Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma. J Clin Invest 2014;124(3):1406–17.28. Mikhaylova I.N., Kovalevsky D.A., Morozova L.F. et al. Cancer/testis genes expression in human melanoma cell lines. Melanoma Res 2008;18(5):303–13.Mikhaylova I.N., Kovalevsky D.A., Morozova L.F. et al. Cancer/testis genes expression in human melanoma cell lines. Melanoma Res 2008;18(5):303–13.29. Miracco C., De Nisi M.C., Arcuri F. et al. Macrophage migration inhibitory factor protein and mRNA expression in cutane ous melanocytic tumours. Int J Oncol 2006;28(2):345–52.Miracco C., De Nisi M.C., Arcuri F. et al. Macrophage migration inhibitory factor protein and mRNA expression in cutane ous melanocytic tumours. Int J Oncol 2006;28(2):345–52.30. Рябая О.О., Цыганова И.В., Сидорова Т .А. и др. Влияние активирующих мутаций V600 гена B-RAF на способность клеток меланомы к аутофагии. Cаркомы костей, мягких тканей и опухоли кожи 2013;(3):68–72. [Ryabaya O.O., Tsyganova I.V., Sidorova Т .А. et al. Effect of Activating V600 mutations of the B-RAF gene on the ability of melanoma cells to autophagy. Sarkomy kostey, myagkikh tkaney i opukholi kozhi = Sarkomas of Bones, Soft Tissues and Skin Tumors 2013;(3):68–72. (In Russ.)].Рябая О.О., Цыганова И.В., Сидорова Т .А. и др. Влияние активирующих мутаций V600 гена B-RAF на способность клеток меланомы к аутофагии. Cаркомы костей, мягких тканей и опухоли кожи 2013;(3):68–72. [Ryabaya O.O., Tsyganova I.V., Sidorova Т .А. et al. Effect of Activating V600 mutations of the B-RAF gene on the ability of melanoma cells to autophagy. Sarkomy kostey, myagkikh tkaney i opukholi kozhi = Sarkomas of Bones, Soft Tissues and Skin Tumors 2013;(3):68–72. (In Russ.)].31. Kimura T., Takabatake Y., Takahashi A., Isaka Y. Chloroquine in cancer therapy: a double-edged sword of autophagy. Cancer Res 2013;73(1):3–7.Kimura T., Takabatake Y., Takahashi A., Isaka Y. Chloroquine in cancer therapy: a double-edged sword of autophagy. Cancer Res 2013;73(1):3–7.32. Diez H., Benitez M.J., Fernandez S. et al. Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation. Biochim Biophys Acta 2016;1863(11):2574–83.Diez H., Benitez M.J., Fernandez S. et al. Class I PI3-kinase or Akt inhibition do not impair axonal polarization, but slow down axonal elongation. Biochim Biophys Acta 2016;1863(11):2574–83.33. Klionsky D.J., Abdelmohsen K., Abe A. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edn). Autophagy 2016;12(1):1–222.Klionsky D.J., Abdelmohsen K., Abe A. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edn). Autophagy 2016;12(1):1–222.34. Mathew R., Karp C.M., Beaudoin B. et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009;137(6):1062–75.Mathew R., Karp C.M., Beaudoin B. et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell 2009;137(6):1062–75.35. Pankiv S., Clausen T.H., Lamark T. et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007;282(33):24131–45.Pankiv S., Clausen T.H., Lamark T. et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 2007;282(33):24131–45.36. Amaravadi R., Kimmelman A.C., White E. Recent insights into the function of autophagy in cancer. Genes Dev 2016;30(17):1913–30.Amaravadi R., Kimmelman A.C., White E. Recent insights into the function of autophagy in cancer. Genes Dev 2016;30(17):1913–30.