Sains Malaysiana 53(3)(2024): 667-679

http://doi.org/10.17576/jsm-2024-5303-14

SF1: A Standardised Fraction of Clinacanthus nutansthat Inhibits the Stemness Properties of Cancer Stem-Like Cells Derived from Cervical Cancer

(SF1: Fraksi Piawaian Clinacanthus nutans yang Merencat Sifat Stem Sel Menyerupai Sel Stem Kanser Diperolehi daripada Kanser Serviks)

FARIDAH ISMAIL^1,2, YUSMAZURA ZAKARIA^1,*, MUHAMMAD LOKMAN MD ISA³, NIK FAKHURUDDIN NIK HASSAN⁴ & TAN SUAT CHENG¹

¹Biomedicine Program, School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, 16150 Kota Bharu, Kelantan, Malaysia

²Department of Basic Medical Sciences, Kulliyyah of Medicine, International Islamic University of Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

³Department of Basic Medical Sciences, Kulliyyah of Nursing, International Islamic University of Malaysia, Jalan Sultan Ahmad Shah, Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia

⁴Forensic Science Program, School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, 16150 Kota Bharu, Kelantan, Malaysia

Diserahkan: 24 Julai2023/Diterima: 21 Februari2024

Abstract

Cancer stem cells (CSCs) are a small population of tumour cells that are responsible for tumour initiation, metastases, recurrence, and resistance to conventional therapy. Hence, targeting CSCs is crucial in the fight against cancer. SF1, a standardised fraction from Clinacanthus nutans leaf extract, has been reported to exhibit potent and selective antineoplastic effects against cervical cancer cells. In this study, the potential of SF1 to inhibit the stemness of cervical cancer stem-like cells has been evaluated. SF1 extraction was carried out using the dry column vacuum chromatography technique. SiHa cell lines were cultured as spheres in CSC-conditioned medium (cervospheres), and the IC₅₀ of SF1 against cervospheres was determined using the OZBlue Cell Viability Kit. The effects of SF1 on the cervosphere’s stemness markers, including CD49f, CK17, SOX2, OCT4, and NANOG, were assessed using a flow cytometry assay. Self-renewal inhibition and anti-tumorigenesis effects of SF1 in cervospheres were evaluated using a sphere formation assay and a xenograft mouse model. The present study shows that SF1 treatment at an IC₅₀ of 17.07 µg/mL inhibited the proliferation, self-renewal, and tumorigenic capacity of SiHa cervospheres in vitro and in vivo. A decrease in the expressions of CK17, SOX2, CD49f, and OCT4 in cervical CSCs indicated that SF1’s inhibitory effects were also associated with the suppression of stemness markers. These results suggest that SF1 possesses an antitumor effect against cervical CSCs and may be regarded as a promising approach to the development of targeted anticancer agents for cervical cancer.

Keywords: Anticancer; cancer stem cells; cervical cancer; Clinacanthus nutans; stemness

Abstrak

Sel stem kanser adalah populasi kecil daripada keseluruhan sel tumor yang bertanggungjawab mencetus pertumbuhan awal tumor, perebakan, pengulangan dan kerintangan terhadap rawatan konvensional. Oleh itu, sasaran rawatan terhadap sel stem kanser adalah penting bagi memerangi kanser. SF1, fraksi yang diseragamkan daripada ekstrak daun Clinacanthus nutans, dilaporkan menunjukkan kesan anti-kanser yang kuat dan selektif terhadap sel kanser serviks. Dalam kajian ini, potensi SF1 untuk merencatkan ciri stem sel yang menyerupai sel stem kanser serviks telah dikaji. SF1 diekstrak menggunakan teknik kromatografi vakum lajur kering. Sel SiHa telah dikultur sebagai sel sfera di dalam kultur media khusus untuk pembentukan sel stem kanser (sel sfera kanser serviks) dan nilai IC₅₀ SF1 terhadap sel sfera kanser serviks ditentukan menggunakan kit kelangsungan hidup OZBlue. Kesan SF1 pada penanda stem di dalam sel sfera kanser serviks, termasuk CD49f, CK17, SOX2, OCT4 dan NANOG, dianalisis menggunakan ujian flositometri. Perencatan pembaharuan diri dan kesan anti-tumorigenesis SF1 dalam sel sfera kanser serviks dinilai menggunakan ujian pembentukan sel sfera dan model tetikus xenocantum. Kajian ini menunjukkan bahawa rawatan SF1 pada IC₅₀ 17.07 μg/mL merencat proliferasi, pembaharuan diri dan keupayaan tumorigenik sel sfera kanser serviks SiHa in vitro dan in vivo. Kesan perencatan SF1 turut disertai oleh penurunan penanda stem untuk sel stem kanser serviks, seperti yang ditunjukkan oleh penurunan dalam pengekspresan CK17, SOX2, CD49f dan OCT4. Hasil kajian ini menunjukkan bahawa SF1 mempunyai kesan anti-tumor terhadap sel stem kanser serviks dan berpotensi sebagai pendekatan baharu untuk membangunkan agen anti-kanser yang bersasar terhadap kanser serviks.

Kata kunci: Anti-kanser; Clinacanthus nutans; kanser serviks; sel stem kanser; sifat stem

RUJUKAN

Alam, A., Ferdosh, S., Ghafoor, K., Hakim, A., Juraimi, A.S. & Sarker, Z.I. 2016. Clinacanthus nutans: A review of the medicinal uses, pharmacology and phytochemistry. Asian Pacific Journal of Tropical Medicine 9(4): 402-409.

Ammothumkandy, A., Maliekal, T.T., Bose, M.V., Rajkumar, T., Shirley, S., Thejaswini, B., Giri, V.G. & Krishna, S. 2016. CD66 and CD49f expressing cells are associated with distinct neoplastic phenotypes and progression in human cervical cancer. European Journal of Cancer 60: 166-178.

Aramini, B., Masciale, V., Grisendi, G., Bertolini, F., Maur, M., Guaitoli, G., Chrystel, I., Morandi, U., Stella, F., Dominici, M. & Haider, K.H. 2022. Dissecting tumor growth: The role of cancer stem cells in drug resistance and recurrence. Cancers (Basel) 14(4): 976.

Bahmad, H.F., Cheaito, K., Chalhoub, R.M., Hadadeh, O., Monzer, A., Ballout, F., El-Hajj, A., Mukherji, D., Liu, Y.N., Daoud, G. & Abou-Kheir, W. 2018. Sphere-formation assay: Three-dimensional in vitro culturing of prostate cancer stem/Progenitor sphere-forming cells. Frontiers in Oncology 8: 347.

Balasubramaniam, S.D., Balakrishnan, V., Oon, C.E. & Kaur, G. 2019. Key molecular events in cervical cancer development. Medicina (Lithuania) 55(7): 384.

Bhattacharjee, R., Dey, T., Kumar, L., Kar, S., Sarkar, R., Ghorai, M., Malik, S., Jha, N.K., Vellingiri, B., Kesari, K.K., Pérez de la Lastra, J.M. & Dey, A. 2022. Cellular landscaping of cisplatin resistance in cervical cancer. Biomedicine and Pharmacotherapy 153: 113345.

Bigoni-ordóñez, G.D., Ortiz-sánchez, E., Rosendo-chalma, P., Valencia-González, H.A., Aceves, C. & García-Carrancá, A. 2018. Molecular iodine inhibits the expression of stemness markers on cancer stem-like cells of established cell lines derived from cervical cancer. BMC Cancer 18(1): 928.

Borah, A., Raveendran, S., Rochani, A., Maekawa, T. & Kumar, D.S. 2015. Targeting self-renewal pathways in cancer stem cells: Clinical implications for cancer therapy. Oncogenesis 4(11): e177.

Burmeister, C.A., Khan, S.F., Schäfer, G., Mbatani, N., Adams, T., Moodley, J. & Prince, S. 2022. Cervical cancer therapies: Current challenges and future perspectives. Tumour Virus Research 13: 200238.

Chang, J.C. 2016. Role in tumor growth, recurrence, metastasis, and treatment resistance. Medicine 95(S1): S20-S25.

Chen, S.F., Chang, Y.C., Nieh, S., Liu, C.L., Yang, C.Y. & Lin, Y.S. 2012. Nonadhesive culture system as a model of rapid sphere formation with cancer stem cell properties. PLoS ONE 7(2): e31864.

Cortes-Dericks, L., Carboni, G.L., Schmid, R.A. & Karoubi, G. 2010. Putative cancer stem cells in malignant pleural mesothelioma show resistance to cisplatin and pemetrexed. International Journal of Oncology 37(2): 437-444.

Di Fiore, R., Suleiman, S., Drago-Ferrante, R., Subbannayya, Y., Pentimalli, F., Giordano, A., & Calleja-Agius, J. 2022. Cancer stem cells and their possible implications in cervical cancer: A short review. International Journal of Molecular Sciences 23(9): 5167.

Ding, Y., Yu, A.Q., Wang, X.L., Guo, X.R., Yuan, Y.H. & Li, D.S. 2016. Forced expression of Nanog with mRNA synthesized in vitro to evaluate the malignancy of HeLa cells through acquiring cancer stem cell phenotypes. Oncology Reports 35(5): 2643-2650.

Feng, D., Peng, C., Li, C., Zhou, Y., Li, M., Ling, B., Wei, H. & Tian, Z. 2009. Identification and characterization of cancer stem-like cells from primary carcinoma of the cervix uteri. Oncology Reports 22(5): 1129-1134.

Fong, S.Y., Piva, T., Dekiwadia, C., Urban, S. & Huynh, T. 2016. Comparison of cytotoxicity between extracts of Clinacanthus nutans (Burm. f.) Lindau leaves from different locations and the induction of apoptosis by the crude methanol leaf extract in D24 human melanoma cells. BMC Complementary and Alternative Medicine 16: 368.

George, I.A., Chauhan, R., Dhawale, R.E., Iyer, R., Limaye, S., Sankaranarayanan, R., Venkataramanan, R. & Kumar, P. 2022. Insights into therapy resistance in cervical cancer. Advances in Cancer Biology – Metastasis 6: 100074.

Huang, D., Guo, W., Gao, J., Chen, J. & Olatunji, J.O. 2015. Clinacanthus nutans (Burm. f.) Lindau ethanol extract inhibits hepatoma in mice through upregulation of the immune response.  Molecules 20(9): 17405-17428.

Huang, R. & Rofstad, E.K. 2017. Cancer stem cells (CSCs), cervical CSCs and targeted therapies. Oncotarget 8(21): 35351-35367.

Ishiguro, T., Ohata, H., Sato, A., Yamawaki, K., Enomoto, T. & Okamoto, K. 2017. Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications. Cancer Science 108(3): 283-289.

Kim, B.W., Cho, H., Choi, C.H., Ylaya, K., Chung, J.Y., Kim, J.H. & Hewitt, S.M. 2015. Clinical significance of OCT4 and SOX2 protein expression in cervical cancer. BMC Cancer 35(5): 2643-2650.

Lee, C.H., Yu, C.C., Wang, B.Y. & Chang, W.W. 2016. Tumorsphere as an effective in vitro platform for screening anti-cancer stem cell drugs. Oncotarget 7(2): 1215-1226.

Li, L. & Neaves, W.B. 2006. Normal stem cells and cancer stem cells: The niche matters. Cancer Research 66(9): 4553-4557.

Liu, H., Zhang, W., Jia, Y., Yu, Q., Grau, G.E., Peng, L., Ran, Y., Yang, Z., Deng, H. & Lou, J.  2013. Single-cell clones of liver cancer stem cells have the potential of differentiating into different types of tumor cells. Cell Death & Disease 4(10): e857.

López, J., Poitevin, A., Mendoza-Martínez, V., Pérez-Plasencia, C. & García-Carrancá, A. 2012. Cancer-initiating cells derived from established cervical cell lines exhibit stem-cell markers and increased radioresistance. BMC Cancer 12: 48.

Martens, J.E., Arends, J., Van der Linden, P.J, De Boer, B.A. & Helmerhorst, T.J. 2004. Cytokeratin 17 and p63 are markers of the HPV target cell, the cervical stem cell. Anticancer Research 24(2B): 771-775.

Messerschmidt, J.L., Bhattacharya, P., Messerschmidt, G.L. 2017. Cancer clonal theory, immune escape, and their evolving roles in cancer multi-agent therapeutics. Curr. Oncol. Rep. 19(10): 66.

Mitra, S.K., Misra, R.K., Varshney, B., Kumari, A. & Rai, D. 2017. Role of p16, Ki67 and CK17 in differentiating benign lesions, cervical intraepithelial neoplasia (CIN) and atypical immature squamous metaplasia (AIM) of uterine cervix. Tropical Journal of Pathology and Microbiology 3(1): 13-19.

Mondal, A., Gandhi, A., Fimognari, C., Atanasov, A.G. & Bishayee, A. 2019. Alkaloids for cancer prevention and therapy: Current progress and future perspectives. European Journal of Pharmacology 858: 172472.

Ng, P.Y., Chye, S.M., Ng, C.H., Koh, R.Y., Tiong, Y.L., Pui, L.P., Tan, Y.H., Siew, C., Lim, Y. & Ng, K.Y. 2017. Clinacanthus nutans hexane extracts induce apoptosis through a caspase-dependent pathway in human cancer cell lines. Asian Pacific Journal of Cancer Prevention 18(4): 917-926.

Organista-Nava, J., Gómez-Gómez, Y., Garibay-Cerdenares, O.L., Leyva-Vázquez, M.A. & Illades-Aguiar, B. 2019. Cervical cancer stem cell-associated genes: Prognostic implications in cervical cancer. Oncology Letters 18(1): 7-14.

Ortiz-sánchez, E., Santiago-López, L., Cruz-Domínguez, V.B., Toledo-Guzmán, M.E., Hernández-Cueto, D. & Muñiz-Hernández, S. 2016. Characterization of cervical cancer stem cell-like cells: Phenotyping, stemness, and human papilloma virus co-receptor expression. Oncotarget 7(22): 31943-31954.

Pandrangi, S.L., Chittineedi, P., Chalumuri, S.S., Meena, A.S., Neira Mosquera, J.A., Sánchez Llaguno, S.N., Pamuru, R.R., Mohiddin, G.J. & Mohammad, A. 2022. Role of intracellular iron in switching apoptosis to ferroptosis to target therapy-resistant cancer stem cells. Molecules 27(9): 3011.

Rositch, A.F., Levinson, K., Suneja, G., Monterosso, A., Schymura, M.J., Mcneel, T.S., Horner, M.J., Engels, E. & Shiels, M.S. 2022. Epidemiology of cervical adenocarcinoma and squamous cell carcinoma among women living with human immunodeficiency virus compared with the general population in the United States. Clinical Infectious Diseases 74(5): 814-820.

Shen, L., Huang, X., Xie, X., Su, J., Yuan, J. & Chen, X. 2014. High expression of SOX2 and OCT4 indicates radiation resistance and an independent negative prognosis in cervical squamous cell carcinoma. Journal of Histochemistry and Cytochemistry 62(7): 499-509.

Singh, D., Vignat, J., Lorenzoni, V., Eslahi, M., Ginsburg, O., Lauby-Secretan, B., Arbyn, M., Basu, P., Bray, F. & Vaccarella, S. 2023. Global estimates of incidence and mortality of cervical cancer in 2020: A baseline analysis of the WHO global cervical cancer elimination initiative. The Lancet Global Health 11(2): e197-e206.

Sun, Q., Wang, L., Zhang, C., Hong, Z. & Han, Z. 2022. Cervical cancer heterogeneity: A constant battle against viruses and drugs. Biomarker Research 10(1): 85.

Swain, N., Thakur, M., Pathak, J. & Swain, B. 2020. SOX2, OCT4 and NANOG: The core embryonic stem cell pluripotency regulators in oral carcinogenesis. Journal of Oral and Maxillofacial Pathology 24(2): 368-373.

Wang, H., Paczulla, A.M., Konantz, M. & Lengerke, C. 2018. In vitro tumorigenic assay: The tumor spheres assay. Methods in Molecular Biology 1692: 77-87.

Wu, L., Han, L., Zhou, C., Wei, W., Chen, X., Yi, H., Wu, X., Bai, X., Guo, S., Yu, Y., Liang, L. & Wang, W. 2017. TGF-β1-induced CK17 enhances cancer stem cell-like properties rather than EMT in promoting cervical cancer metastasis via the ERK1/2-MZF1 signaling pathway. FEBS Journal 284(18): 3000-3017.

Yahaya, R., Dash, G.K., Abdullah, M.S. & Mathews, A. 2015. Clinacanthus nutans (burm. F.) Lindau: An useful medicinal plant of South-East Asia. International Journal of Pharmacognosy and Phytochemical Research 7(6): 1244-1250.

Yong, Y.K., Tan, J.J., Teh, S.S., Mah, S.H., Ee, G.C.L., Chiong, H.S. & Ahmad, Z. 2013. Clinacanthus nutans extracts are antioxidant with antiproliferative effect on cultured human cancer cell lines. Evidence-Based Complementary and Alternative Medicine 2013: 462751.

Zainuddin, N.A.S.N., Muhammad, H., Nik Hassan, N.F., Othman, N.H. & Zakaria, Y. 2020. Clinacanthus nutans standardized fraction arrested SiHa cells at G1/S and induced apoptosis via upregulation of p53. Journal of Pharmacy and Bioallied Sciences 12(2): 768-776.

Zainuddin, N.A.S.N., Hassan, N.F.N., Zakaria, Y., Muhammad, H. & Othman, N.H. 2019. Semi-purified fraction of Clinacanthus nutans induced apoptosis in human cervical cancer, SiHa cells via up-regulation of Bax and down-regulation of Bcl-2. Sains Malaysiana 48(9): 1997-2006.

Zhang, S.L., Wang, Y.S., Zhou, T., Yu, X.W., Wei, Z.T. & Li, Y.L. 2012. Isolation and characterization of cancer stem cells from cervical cancer HeLa cells. Cytotechnology 64(4): 477-484.

*Pengarang untuk surat-menyurat; email: yusmazura@usm.my