Promotion of Cell Cycle Arrest and Inhibition of Anchorage-independent Growth of Cervical Cancer HeLa Cells by Ethanol Extract of Croton kongensis
Main Article Content
Abstract
Extracts from Croton kongensis (C. kongensis) exhibit anticancer activities on various cancers. However, there is no research conducted to investigate the effects of C. kongensis extracts on cervical cancer as well as on zebrafish. In this study, we demonstrated that C. kongensis ethanol extract expressed high toxicity to cervical cancer HeLa cells with an IC50 dose of 20.4 µg/mL and to zebrafish embryos with malformations, lethality and hatching inhibition at 72-hpf at the effective dose of 125 µg/mL. Interestingly, treatment with C. kongensis ethanol extract caused cell cycle arrest at the G2 phase. Particularly, percentages of C. kongensis ethanol extract-treated cells in G1, S, G2/M were 70%, 6% and 23%, while percentages of control cells in G1, S, G2/M were 65%, 15% and 18%, respectively. Consistent with cell cycle arrest, the expressions of CDKN1A, CDNK2A and p53 in C. kongensis ethanol extract-treated cells were up-regulated 2.0-, 1.65- and 1.8-fold, respectively. Significantly, treatment with C. kongensis ethanol extract inhibited anchorage-independent growth of HeLa cells; the number of colonies formed in soft-agar of C. kongensis ethanol extract-treated cells was only one-fourth of that of control cells. Overall, we suggest that C. kongensis ethanol extract should be used as a traditional medicine for treatment of cervical cancer.
Keywords:
Croton kongensis, HeLa cells, zebrafish, cell cycle arrest, anchorage-independent growth.
References
[1] P. T. Thuong, T. T. Dao, T. H. Pham, P. H. Nguyen, T. V. Le, K. Y. Lee, W. K. Oh. Crotonkinensins A and B, Diterpenoids from the Vietnamese Medicinal Plant Croton Tonkinensis, Journal of Natural Products, Vol. 72, No. 11, 2009, pp.2040-2042.
[2] P. T. Thuong, T. H. Pham, T. V. Le, T. T. Dao, T. T. Dang, Q. T. Nguyen, W. K. Oh. Symmetric Dimers of Ent-kaurane Diterpenoids with Cytotoxic Activity from Croton Tonkinensis, Bioorganic and Medicinal Chemistry Letters, Vol. 22, No. 2, 2012, pp. 1122-1124.
[3] N. H. Nguyen, H. T. Vu, N. D. Le, T. D. Nguyen, H. X. Duong, H. D. Tran, Molecular Identification and Evaluation of the Genetic Diversity of Dendrobium Species Collected in Southern Vietnam, Biology, Vol. 9, No. 4, 2020, pp. 76.
[4] T. M. H. Nguyen, H. L. Le, T. T. Ha, B. H. Bui, N. T. Le, V. H. Nguyen, T. V. A. Nguyen. Inhibitory Effect on Human Platelet Aggregation and Coagulation and Antioxidant Activity of C. Edulis Ker Gawl Rhizome and Its Secondary Metabolites, Journal of Ethnopharmacology, Vol. 263, 2020,
pp. 113136.
[5] WHO, Cancer Today, IARC CancerBase No. 15. Edited by J. Ferlay, M. Ervik, F. Lam, M. Colombet, L. Mery, M. Piñeros, A. Znaor, I. Soerjomataram, F. Bray, 2018.
[6] D. Stelzle, L. F. Tanaka, K. K. Lee, A. K. Ibrahim, I. Baussano, A. S. V. Shah, D. A. McAllister, S. L. Gottlieb, S. J. Klug, A. S. Winkler et al., Estimates of the Global Burden of Cervical Cancer Associated with HIV, Lancet Global Health, Vol. 9, No. 2, 2021, pp. e161-e169.
[7] P. C. Kuo, Y. C. Shen, M. L. Yang, S. H. Wang, T. D. Thang, N. X. Dung, P. C. Chiang, K. H. Lee, E. J. Lee, T. S. Wu, Crotonkinins A and B and Related Diterpenoids from Croton Tonkinensis as Anti-inflammatory and Antitumor Agents, Journal of Natural Products, Vol. 70, No. 12, 2007, pp. 1906-1909.
[8] P. C. Kuo, M. L. Yang, T. L. Hwang, Y. Y. Lai, Y. C. Li, T. D. Thang, T. S. Wu. Anti-inflammatory Diterpenoids from Croton Tonkinensis, Journal of Natural Products, Vol. 76, No. 2, 2013, pp. 230-236.
[9] Y. H. Sul, M. S. Lee, E. Y. Cha, P. T. Thuong, N. M. Khoi, I. S. Song, An Ent-kaurane Diterpenoid from Croton Tonkinensis Induces Apoptosis by Regulating AMP-activated Protein Kinase in SK-HEP1 Human Hepatocellular Carcinoma Cells, Biological and Pharmaceutical Bulletin, Vol. 36, No. 1, 2013, pp. 158-164.
[10] P. T. Thuong, N. M. Khoi, S. Ohta, S. Shiota, H. Kanta, K. Takeuchi, F. Ito. Ent-kaurane Diterpenoids from Croton Tonkinensis Induce Apoptosis in Colorectal Cancer Cells Through the Phosphorylation of JNK Mediated by Reactive Oxygen Species and Dual-specificity JNK Kinase MKK4, Anticancer Agents in Medicinal Chemistry, Vol. 14, No. 7, 2014, pp. 1051-1061.
[11] M. Q. Pham, A. L. Iscache, Q. L. Pham, J. E. Gairin, Cytotoxic, Apoptotic, and Sensitization Properties of Ent-kaurane-type Diterpenoids from Croton Tonkinensis Gagnep on Human Liver Cancer HepG2 and Hep3b Cell Lines, Fundamental and Clinical Pharmacology, Vol. 30, No. 2, 2016, pp. 137-146.
[12] OECD, Guidelines for the Testing of Chemicals, Section 4, Test No. 423: Acute Oral Toxicity - Acute Toxic Class Method, OECD Publishing, 2002.
[13] K. Ando, S. Fukuhara, N. Izumi, H. Nakajima, H. Fukui, R. N. Kelsh, N. Mochizuki, Clarification of Mural Cell Coverage of Vascular Endothelial Cells by Live Imaging of Zebrafish, Development, Vol. 143, No. 8, 2016, pp. 1328-1339.
[14] N. T. B. Loan, K. T. Kien, N. L Thanh et al., Toxicity and Anti-Proliferative Properties of Anisomeles indica Ethanol Extract on Cervical Cancer HeLa Cells and Zebrafish Embryos, Life, Vol. 11, No. 3, 2021, pp. 257.
[15] G. Soman, X. Yang, H. Jiang, S. Giardina, V. Vyas, G. Mitra, J. Yovandich, S. P. Creekmore, T. A. Waldmann, O. Quiñones, W. G. Alvord, MTS Dye Based Colorimetric CTLL-2 Cell Proliferation Assay for Product Release and Stability Monitoring of Interleukin-15: Assay Qualification, Standardization and Statistical Analysis, Journal of Immunological Methods, Vol. 348, No. 1-2, 2009, pp. 83-94.
[16] K. H. Kim, J. M. Sederstrom, Assaying Cell Cycle Status Using Flow Cytometry, Current Protocols in Molecular Biology, Vol. 111, 2015, pp. 28.6.1-28.6.11.
[17] N. D. Thang, I. Yajima, K. Y. Kumasaka, S. Ohnuma, T. Yanagishita, R. Hayashi, H. U. Shekhar, D. Watanabe, M. Kato, Barium Promotes Anchorage-independent Growth and Invasion of Human HaCaT Keratinocytes Via Activation of c-SRC Kinase, PLoS One, Vol. 6, No. 10, 2011, pp. e25636.
[18] N. Ohgami, O. Yamanoshita, N. D. Thang, I. Yajima, C. Nakano, W. Wenting, S. Ohnuma, M. Kato, Carcinogenic Risk of Chromium, Copper and Arsenic in CCA-treated Wood, Environtal Pollution, Vol. 206, 2015, pp. 456-460.
[19] A. J. Levine, The Cellular Gatekeeper for Growth and Division, Cell, Vol. 88, 1997, pp. 323.
[20] P. G. G. Hemmati, B. Normand, J. Gillissen,. Wendt, B. Dörken, P. T. Daniel, Cooperative Effect of p21Cip1/WAF-1 and 14-3-3sigma on Cell Cycle Arrest and Apoptosis Induction by p14ARF, Oncogene, Vol. 27, 2008, pp. 6707-6719.
[21] C. Romagosa, S. Simonetti, L. López-Vicente, A. Mazo, M. E. Lleonart, J. Castellvi, S. Ramon, Cajal, p16Ink4a Overexpression in Cancer: a Tumor Suppressor Gene Associated with Senescence and High-grade Tumors, Oncogene, Vol. 30, 2011, pp. 2087-2097.
[22] Z. Li, J. Zhao, Y. Du, H. R. Park, S. Y. Sun, L. Bernal-Mizrachi, A. Aitken, F. R. Khuri, H. Fu, Down-regulation of 14-3-3zeta Suppresses Anchorage-independent Growth of Lung Cancer Cells Through Anoikis Activation, Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 1, 2008, pp. 162-167.
[23] F. Weinberg, R. Hamanaka, W. W. Wheaton, S. Weinberg, J. Joseph, M. Lopez, B. Kalyanaraman, G. M. Mutlu, G. R. Budinger, N. S. Chandel, Mitochondrial Metabolism and ROS Generation are Essential for Kras-mediated Tumorigenicity, Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 19, 2010, pp. 8788-8793.
[2] P. T. Thuong, T. H. Pham, T. V. Le, T. T. Dao, T. T. Dang, Q. T. Nguyen, W. K. Oh. Symmetric Dimers of Ent-kaurane Diterpenoids with Cytotoxic Activity from Croton Tonkinensis, Bioorganic and Medicinal Chemistry Letters, Vol. 22, No. 2, 2012, pp. 1122-1124.
[3] N. H. Nguyen, H. T. Vu, N. D. Le, T. D. Nguyen, H. X. Duong, H. D. Tran, Molecular Identification and Evaluation of the Genetic Diversity of Dendrobium Species Collected in Southern Vietnam, Biology, Vol. 9, No. 4, 2020, pp. 76.
[4] T. M. H. Nguyen, H. L. Le, T. T. Ha, B. H. Bui, N. T. Le, V. H. Nguyen, T. V. A. Nguyen. Inhibitory Effect on Human Platelet Aggregation and Coagulation and Antioxidant Activity of C. Edulis Ker Gawl Rhizome and Its Secondary Metabolites, Journal of Ethnopharmacology, Vol. 263, 2020,
pp. 113136.
[5] WHO, Cancer Today, IARC CancerBase No. 15. Edited by J. Ferlay, M. Ervik, F. Lam, M. Colombet, L. Mery, M. Piñeros, A. Znaor, I. Soerjomataram, F. Bray, 2018.
[6] D. Stelzle, L. F. Tanaka, K. K. Lee, A. K. Ibrahim, I. Baussano, A. S. V. Shah, D. A. McAllister, S. L. Gottlieb, S. J. Klug, A. S. Winkler et al., Estimates of the Global Burden of Cervical Cancer Associated with HIV, Lancet Global Health, Vol. 9, No. 2, 2021, pp. e161-e169.
[7] P. C. Kuo, Y. C. Shen, M. L. Yang, S. H. Wang, T. D. Thang, N. X. Dung, P. C. Chiang, K. H. Lee, E. J. Lee, T. S. Wu, Crotonkinins A and B and Related Diterpenoids from Croton Tonkinensis as Anti-inflammatory and Antitumor Agents, Journal of Natural Products, Vol. 70, No. 12, 2007, pp. 1906-1909.
[8] P. C. Kuo, M. L. Yang, T. L. Hwang, Y. Y. Lai, Y. C. Li, T. D. Thang, T. S. Wu. Anti-inflammatory Diterpenoids from Croton Tonkinensis, Journal of Natural Products, Vol. 76, No. 2, 2013, pp. 230-236.
[9] Y. H. Sul, M. S. Lee, E. Y. Cha, P. T. Thuong, N. M. Khoi, I. S. Song, An Ent-kaurane Diterpenoid from Croton Tonkinensis Induces Apoptosis by Regulating AMP-activated Protein Kinase in SK-HEP1 Human Hepatocellular Carcinoma Cells, Biological and Pharmaceutical Bulletin, Vol. 36, No. 1, 2013, pp. 158-164.
[10] P. T. Thuong, N. M. Khoi, S. Ohta, S. Shiota, H. Kanta, K. Takeuchi, F. Ito. Ent-kaurane Diterpenoids from Croton Tonkinensis Induce Apoptosis in Colorectal Cancer Cells Through the Phosphorylation of JNK Mediated by Reactive Oxygen Species and Dual-specificity JNK Kinase MKK4, Anticancer Agents in Medicinal Chemistry, Vol. 14, No. 7, 2014, pp. 1051-1061.
[11] M. Q. Pham, A. L. Iscache, Q. L. Pham, J. E. Gairin, Cytotoxic, Apoptotic, and Sensitization Properties of Ent-kaurane-type Diterpenoids from Croton Tonkinensis Gagnep on Human Liver Cancer HepG2 and Hep3b Cell Lines, Fundamental and Clinical Pharmacology, Vol. 30, No. 2, 2016, pp. 137-146.
[12] OECD, Guidelines for the Testing of Chemicals, Section 4, Test No. 423: Acute Oral Toxicity - Acute Toxic Class Method, OECD Publishing, 2002.
[13] K. Ando, S. Fukuhara, N. Izumi, H. Nakajima, H. Fukui, R. N. Kelsh, N. Mochizuki, Clarification of Mural Cell Coverage of Vascular Endothelial Cells by Live Imaging of Zebrafish, Development, Vol. 143, No. 8, 2016, pp. 1328-1339.
[14] N. T. B. Loan, K. T. Kien, N. L Thanh et al., Toxicity and Anti-Proliferative Properties of Anisomeles indica Ethanol Extract on Cervical Cancer HeLa Cells and Zebrafish Embryos, Life, Vol. 11, No. 3, 2021, pp. 257.
[15] G. Soman, X. Yang, H. Jiang, S. Giardina, V. Vyas, G. Mitra, J. Yovandich, S. P. Creekmore, T. A. Waldmann, O. Quiñones, W. G. Alvord, MTS Dye Based Colorimetric CTLL-2 Cell Proliferation Assay for Product Release and Stability Monitoring of Interleukin-15: Assay Qualification, Standardization and Statistical Analysis, Journal of Immunological Methods, Vol. 348, No. 1-2, 2009, pp. 83-94.
[16] K. H. Kim, J. M. Sederstrom, Assaying Cell Cycle Status Using Flow Cytometry, Current Protocols in Molecular Biology, Vol. 111, 2015, pp. 28.6.1-28.6.11.
[17] N. D. Thang, I. Yajima, K. Y. Kumasaka, S. Ohnuma, T. Yanagishita, R. Hayashi, H. U. Shekhar, D. Watanabe, M. Kato, Barium Promotes Anchorage-independent Growth and Invasion of Human HaCaT Keratinocytes Via Activation of c-SRC Kinase, PLoS One, Vol. 6, No. 10, 2011, pp. e25636.
[18] N. Ohgami, O. Yamanoshita, N. D. Thang, I. Yajima, C. Nakano, W. Wenting, S. Ohnuma, M. Kato, Carcinogenic Risk of Chromium, Copper and Arsenic in CCA-treated Wood, Environtal Pollution, Vol. 206, 2015, pp. 456-460.
[19] A. J. Levine, The Cellular Gatekeeper for Growth and Division, Cell, Vol. 88, 1997, pp. 323.
[20] P. G. G. Hemmati, B. Normand, J. Gillissen,. Wendt, B. Dörken, P. T. Daniel, Cooperative Effect of p21Cip1/WAF-1 and 14-3-3sigma on Cell Cycle Arrest and Apoptosis Induction by p14ARF, Oncogene, Vol. 27, 2008, pp. 6707-6719.
[21] C. Romagosa, S. Simonetti, L. López-Vicente, A. Mazo, M. E. Lleonart, J. Castellvi, S. Ramon, Cajal, p16Ink4a Overexpression in Cancer: a Tumor Suppressor Gene Associated with Senescence and High-grade Tumors, Oncogene, Vol. 30, 2011, pp. 2087-2097.
[22] Z. Li, J. Zhao, Y. Du, H. R. Park, S. Y. Sun, L. Bernal-Mizrachi, A. Aitken, F. R. Khuri, H. Fu, Down-regulation of 14-3-3zeta Suppresses Anchorage-independent Growth of Lung Cancer Cells Through Anoikis Activation, Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 1, 2008, pp. 162-167.
[23] F. Weinberg, R. Hamanaka, W. W. Wheaton, S. Weinberg, J. Joseph, M. Lopez, B. Kalyanaraman, G. M. Mutlu, G. R. Budinger, N. S. Chandel, Mitochondrial Metabolism and ROS Generation are Essential for Kras-mediated Tumorigenicity, Proceedings of the National Academy of Sciences of the United States of America, Vol. 107, No. 19, 2010, pp. 8788-8793.