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

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 (IC 50 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. 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 separapenulenan, SF1 daripada C. nutans terhadap kanser serviks. Penfraksian 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 IC 50 = 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 apoptosi...

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

“…The extraction was performed in accordance with the published protocol (Zainuddin et al 2019). Briefly, the collected leaves were dried and ground to a coarse powder.…”

“…Prior to cell treatment, the SF1 was diluted in DMSO (Nacalai Tesque, Japan) and followed by a serial dilution ranging from 0.2 to 100 µg/mL. The selection of these concentrations was based on the cytotoxicity studies previously conducted for SF1 (Zainuddin et al 2019). Cisplatin (Sigma-Aldrich, USA) was used as a positive control, whereas DMSO was used as a negative (vehicle) control with a concentration less than 0.1%.…”

“…SF1 is a semi-purified fraction isolated from the leaves of C. nutans using bioassay-guided fractionation. The fraction has demonstrated potent anticancer effects on cervical cancer cells by causing DNA damage, cell cycle arrest, and triggering apoptosis (Zainuddin et al 2019). Alkaloids with functional group amines emerged as the dominant phytochemical class in the analysis of SF1 using Fourier transform infrared spectroscopy (FTIR) and liquid chromatography-mass spectrometry (LC-MS), followed by alkanes, alkenes, esters, and aromatics (Zainuddin et al 2020).…”

“…Previous studies have demonstrated that SF1 has potent inhibitory effects against cervical cancer cell lines and the proposed mechanisms (Zainuddin et al 2020(Zainuddin et al , 2019. In an attempt to bring this research forward, the effect of the SF1 fraction in CCSCs was further evaluated.…”

“…This standardised fraction was found to induce cervical cancer cell death via apoptosis and cell growth inhibition at the G1 phase checkpoint. In particular, SF1 has shown a selective inhibitory effect on cancer cells while sparing normal cells (Zainuddin et al 2020(Zainuddin et al , 2019. Therefore, the potential of SF1 as a safe and effective anticancer agent is promising.…”