Sains Malaysiana 48(9)(2019): 1997–2006

http://dx.doi.org/10.17576/jsm-2019-4809-21

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

(Fraksi Separa Tulen Clinacanthus nutans Teraruh Apoptosis pada Kanser Pangkal Rahim Manusia, Sel-sel SiHa melalui Kawal Atur-atas Bax dan Kawal Atur-bawah Bcl-2)

NIK AINA SYAZANA NIK ZAINUDDIN¹, HUSSIN MUHAMMAD², NIK FAKHURUDDIN NIK HASSAN¹, NOR HAYATI OTHMAN³ & YUSMAZURA ZAKARIA¹*

¹School of Health Sciences, USM Health Campus, 16150 Kota Bharu, Kelantan Darul Naim, Malaysia

²Herbal Medicine Research Centre, Institute for Medical Research (IMR), 50588 Kuala Lumpur, Federal Territory, Malaysia

³School of Medical Sciences, USM Health Campus, 16150 Kota Bharu, Kelantan Darul Naim, Malaysia

Received: 29 October 2018/Accepted: 22 July 2019

ABSTRACT

Cervical cancer is a leading cause of death in women, worldwide, and second in Malaysia. Nowadays, modern cancer treatment comes with negative and adverse side effects to the patients. Traditional medicinal plants such as Clinacanthus nutans has been locally recognized for its medicinal properties and was claimed for cancer treatment. According to our pilot study, a fraction of C. nutans has shown a potent inhibition on human cervical cancer cells, SiHa, in vitro. Hence, this study aimed to investigate the underlying anticancer mechanism of a semi-purified fraction (SF1) of C. nutans towards cervical cancer cells. Bioassay guided fractionation and further purification was carried out. All collected fractions of C. nutans were examined for cytotoxicity activity towards HeLa, SiHa and NIH cells by MTT Assay. SF1 exhibited better cytotoxic activity with best growth inhibition against SiHa cells (IC50 value = 9.60 μg/mL). Cytoselective effects were shown as no cytotoxicity observed in the treated normal NIH cells. SF1 induced early apoptosis event in SiHa cells as resulted by FITC-Annexin V/propidium iodide staining by flowcytometric analysis. The protein expression by flowcytometry using specific antibody conjugated fluorescent dye showed up-regulation of Bax and down-regulation Bcl-2 as well as increment of cytochrome C levels upon treatment with SF1. In conclusion, the findings suggested that SF1 demonstrated antiproliferative effects and induced apoptosis in SiHa cells through up-regulation of Bax and down-regulation of Bcl-2 via mitochondrial-dependent pathway.

Keywords: Apoptosis; cervical cancer; Clinacanthus nutans

ABSTRAK

Kanser pangkal rahim adalah penyebab utama kematian wanita di seluruh dunia dan kedua di Malaysia. Pada masa ini, rawatan moden kanser banyak memberi kesan negatif kepada pesakit. Tumbuhan ubatan tradisi seperti Clinacanthus nutans telah dikenal pasti dan dipercayai oleh penduduk tempatan sebagai alternatif kepada rawatan kanser. Menurut kajian awal kami, satu fraksi daripada C. nutans telah menunjukkan perencatan yang aktif terhadap kanser serviks manusia, secara in vitro. Oleh itu, penyelidikan ini bertujuan untuk mengkaji mekanisme antikanser oleh fraksi separa - penulenan, SF1 daripada C. nutans terhadap kanser serviks. Pemfraksian berpandukan bioaktiviti dan penulenan lanjutan telah dijalankan. Semua fraksi yang terkumpul daripada C. nutans telah diuji untuk aktiviti kesitoksikan terhadap sel HeLa, SiHa dan NIH oleh asai MTT. SF1 menunjukkan aktiviti sitotoksik yang lebih baik dengan antiproliferasi paling berpotensi terhadap sel SiHa (nilai IC50 = 9.60 μg/mL) dan menunjukkan kesan sitomemilih terhadap sel normal, NIH. Kesan apoptosis awal dapat diaruhkan terhadap sel SiHa daripada rawatan SF1 melalui analisis flositometri oleh pewarnaan FITC-Annexin V/propidium iodida. Pengekspresan protein menggunakan antibodi khusus berkonjugasi pendarfluor oleh analisis flositometri juga menunjukkan pengawalaturan Bax dan Bcl-2 serta peningkatan kadar sitokrom C setelah dirawat dengan SF1. Kesimpulannya, SF1 menunjukkan kesan antiproliferatif dan apoptosis yang diaruhkan dalam sel SiHa melalui pengawalaturan Bax dan Bcl-2.

Kata kunci: Apoptosis; Clinacanthus nutans; kanser serviks

REFERENCES

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

Boik, J. 2001. Natural Compounds in Cancer Therapy. Minnesota: Oregon Medical Press.

Bruni, L.B.R.L., Albero, G., Serrano, B., Mena, M., Gómez, D., Muñoz, J., Bosch, F.X. & Sanjosé, S. 2018. Human Papillomavirus and Related Diseases in Asia. ICO/IARC Information Centre on HPV and Cancer (HPV Information Centre), Spain.

Chipuk, J.E. & Green, D.R. 2008. How do BCl-2 proteins induce mitochondrial outer membrane permeabilization? Trends in Cel. Biology 18(4): 57-164.

Ch’ng, Y.S., Tan, C.S., Loh, Y.C., Ahmad, M., Asmawi, M.Z. & Yam, M.F. 2016. Vasorelaxation study and tri-step infrared spectroscopy analysis of Malaysian local herbs. Journal of Pharmacopuncture 19(2): 145-154.

Choudari, A.S., Suryavanshi, S.A. & Kaul-Ghanekar, R. 2013. The aqueous extract of Ficus religiosa induces cell cycle arrest in human cervical cancer cell lines SiHa (HPV-16 positive) and apoptosis in HeLa (HPV-18 positive). PLoS ONE 8(7): e70127.

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.

Ghasemzadeh, A., Nasiri, A., Jaafar, H.Z.E., Baghdadi, A. & Ahmad, I. 2014. Changes in phytochemical synthesis, chalcone synthase activity and pharmaceutical qualities of Sabah Snake Grass (Clinacanthus nutans L.) in relation to plant age. Molecules 19: 17632-17648.

He, S.H., Liu, H.G., Zhou, Y.F. & Yue, Q.F. 2017. Liquiritin (LT) exhibits suppressive effects against the growth of human cervical cancer cells through activating caspase-3 in vitro and xenograft mice in vivo. Biomedicine & Pharmacotherapy 92: 215-228.

Huang, D., Li, Y., Cui, F., Chen, J. & Sun, J. 2016. Purification and characterization of a novel polysaccharide-peptide complex from Clinacanthus nutans Lindau extracts. Carbohydrate Polymers 137: 701-708.

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: 17405-17428.

Indramalar, S. 2015. No women should die from cervical cancer. The Star Online. 16th Jan.

Jaudan, A., Sharma, S., Malek, S.N.A. & Dixit, A. 2018. Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action. PLoS ONE 13(2): 0191523.

JuÈrgensmeier, J.M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D. & Reed, J.C. 1998. Bax directly induces release of cytochrome C from isolated mitochondria. Proceedings of the National Academy of Sciences of the United States of America 95: 4997-5002.

Le, C.F., Kailaivasan, T.H., Sek-Chuen, C., Zunoliza, A., Sui- Kiong, L. & Chee-Mun, F. 2017. Phytosterols isolated from Clinacanthus nutans induce immunosuppressive in murine cells. International Immunopharmacology 44: 203-210.

Ng, P.Y., Soi, M.C., Liew, P.P., Chew, H.N., Yong, H.T., Rhun, Y.K., Crystale, S.Y.L., Khuen, Y.N. & Yee, L.T. 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.

Roslan, S.N.F.M., Yusmazura, Z. & Hasmah, A. 2018. Cytotoxicity of Clinacanthus nutans and mechanism of action of its active fraction towards human cervical cancer cell line, HeLA. Jurnal Sains Kesihatan Malaysia 16(2): 39-50.

Sakdarat, S., Shuyprom, A., Pientong, C., Ekalaksananan, T. & Thongchai, S. 2009. Bioactive constituents from the leaves of Clinacanthus nutans Lindau. Bioorganic & Medicinal Chemistry 17: 1857-1860.

Santin, G., Piccolini, V.M., Veneroni, P., Barni, S., Bernocchi, G. & Bottone, M.G. 2011. Different patterns of apoptosis in response to cisplatin in B50 neuroblastoma rat cells. Histology and Histopathology 26: 831-842.

Sulaiman, I.S.C., Basri, M., Chan, K.W., Ashari, S.E., Masoumi, H.R.F. & Ismail, M. 2015. In vitro antioxidant, cytotoxic and phytochemical studies of Clinacanthus nutans Lindau leaf extracts. African Journal of Pharmacy and Pharmacology 9(34): 861-874.

Teoh, P.L., Cheng, A.Y.F., Liau, M., Lem, F.F., Kaling, G.P., Chua, F.N. & Cheong, B.E. 2016. Chemical composition and cytotoxic properties of Clinacanthus nutans root extracts Pharmaceutical Biology 55(1): 394-401.

World Health Organization. 2017. http://apps.who.int/ medicinedocs/en/d/Jh2945e/2.1.html. Accessed on 6 June 2017.

Xu, W., Mi, Y., He, P., He, S. & Niu, L. 2017. γ-Tocotrienol inhibits proliferation and induces apoptosis via the mitochondrial pathway in human cervical cancer HeLa cells. Molecules 22: 1292-1299.

Yong, Y.K., Tan, J.J., Teoh, 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.

Zakaria, Y., Yee, L.W. & Hassan, N.F.N. 2017. Anti-cancer effects of Clinacanthus nutans extract towards human cervical cancer cell line, HeLa. Journal of Biomedicine & Clinical Science 2(1): 11-19.

Zazali, K., Abdullah, H. & Jamil, N. 2013. Methanol extract of Oroxylum indicum leaves induces G1/S cell cycle arrest in HeLa cells via p53-mediated pathway. International Journal of Medicinal Plant Research 2(7): 225-237.

Zhen, S., Lu, J.J., Wang, L.J., Sun, X.M., Zhang, J.Q., Li, X., Luo, W.J. & Zhao, L. 2016. In vitro and in vivo synergistic therapeutic effect of cisplatin with human papillomavirus16 E6/E7 CRISPR/Cas9 on cervical cancer cell line. Translational Oncology 9(6): 498-504.

*Corresponding author; email: yusmazura@usm.my