PLoS One. 2013 Jul 26;8(7):e70127.

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).

Amit S Choudhari, Snehal A Suryavanshi and Ruchika Kaul-Ghanekar*

Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth University Medical College Campus, Dhankawadi, Pune 411043, India

 

Abstract

Natural products are being extensively explored for their potential to prevent as well as treat cancer due to their ability to target multiple molecular pathways. Ficus religiosa has been shown to exert diverse biological activities including apoptosis in breast cancer cell lines. In the present study, we report the anti-neoplastic potential of aqueous extract of F. religiosa (FRaq) bark in human cervical cancer cell lines, SiHa and HeLa. FRaq altered the growth kinetics of SiHa (HPV-16 positive) and HeLa (HPV-18 positive) cells in a dose-dependent manner. It blocked the cell cycle progression at G1/S phase in SiHa that was characterized by an increase in the expression of p53, p21 and pRb proteins with a simultaneous decrease in the expression of phospho Rb (ppRb) protein. On the other hand, in HeLa, FRaq induced apoptosis through an increase in intracellular Ca(2+) leading to loss of mitochondrial membrane potential, release of cytochrome-c and increase in the expression of caspase-3. Moreover, FRaq reduced the migration as well as invasion capability of both the cervical cancer cell lines accompanied with downregulation of MMP-2 and Her-2 expression. Interestingly, FRaq reduced the expression of viral oncoproteins E6 and E7 in both the cervical cancer cell lines. All these data suggest that F. religiosa could be explored for its chemopreventive potential in cervical cancer.

PMID: 23922932

 

SUPPLEMENT:

Cervical cancer is one of the most common cancers in the women worldwide. Human papilloma virus (HPV) infection is considered to be the main risk factor for the development of cervical cancer, wherein HPV-16 and -18 account for about 70% of the invasive cancers. Conventional therapies taht include radiation as well as chemotherapy oprions, have wide range of side-effects as not only the tumor cells but the normal healthy cells also get killed. Recently, lot of attention is being focused towards identification of new alternative approaches that would reduce morbidity as well as side-effects conferred by conventional treatments.

Traditional medicines have been popular in Asian countries in the management of cancer from time immemorial and these approaches are being increasingly accepted in the rest of the world as complementary and alternative methods. Various scientific studies have suggested the potential of medicinal plants as anti-cancer drug candidates. Our group had previously reported the cytotoxic activity of Cinnamomum zeylanicum in a variety of cancerous cell lines (Singh R et al., 2009). We had also previously elucidated the putative mechanism underlying the anti-neoplastic potential of Cinnamomum cassia (cinnamon) in cervical cancer cell line (Koppikar SJ et al., 2010).

We have previously reported that the aqueous and ethanolic extracts of F. religiosa bark exhibited significant ‘total antioxidant capacity’ and showed appreciable cytotoxicity in cervical cancer cell lines, SiHa and HeLa (Choudhari A S et al, 2011). Ficus religiosa Linn. (Moraceae), commonly known as peepal, is widely cultivated in southeast Asia and has been known for many medicinal properties in traditional system. In the present study, we have evaluated the anti-neoplastic potential of aqueous extract of F. religiosa bark (FRaq) in cervical cancer cells. Our study shows that FRaq downregulated the expression of E6 oncoprotein and simultaneously increased the expression of p53 in both SiHa and HeLa cell lines. The increased p53 expression resulted into cell cycle arrest in SiHa at G1/S phase, characterized by an increase in p21 and pRb with a simultaneous decrease in E7 and phospho Rb (ppRb) expressions. On the other hand, the increased p53 expression resulted into apoptosis in HeLa cells through increase in intracellular Ca2+ leading to loss of mitochondrial membrane potential and increase in cytochrome c and caspase-3 expressions. Moreover, FRaq reduced the migration as well as invasion in both the cervical cancer cells through downregulation of MMP-2 and Her-2 proteins. These results highlighted significant differences in growth regulation of cervical cancer cells by FRaq (Scheme 1).

The observed dichotomy in the regulation of growth in SiHa and HeLa could be due to variation in p53 activation that may decide the fate of a cell to either initiate apoptosis or undergo cell cycle arrest. It has already been reported that low levels of p53 induce cell cycle arrest whereas high levels of p53 induce apoptosis. Our results show that p53 was activated more in HeLa compared to SiHa in response to FRaq treatment, thereby, resulting into apoptosis in the former and cell cycle arrest in the latter. The other reason for the altered response of SiHa and HeLa towards Ficus treatment could be the difference in their genetic make-up that includes their HPV status as well as the viral copy number. For example, SiHa (squamous cell carcinoma) contains around 1–2 integrated copies of HPV 16 genome whereas HeLa (adenocarcinoma) has around 10–50 integrated copies of HPV 18. Moreover, the rate of replication is also different in both the cell types.

Plant polyphenols have recently invited a great deal of attention owing to their chemopreventive properties and F. religiosa is rich in polyphenols. They can modulate the process of carcinogenesis through several mechanisms. Our findings provide a strong basis for further exploration of Ficus religiosa as a potent therapeutic drug for cervical cancer.

 

Acknowledgments

This work was supported by grants from IRSHA, Bharati Vidyapeeth University and Council for Scientific and Industrial Research, Government of India.

 

References:

  1. Choudhari, A.S. et al. (2011) Evaluating the antioxidant potential of aqueous and alcoholic extracts of Ficus religiosa using ORAC assay and assessing their cytotoxic activity in cervical cancer cell lines. Biotechnol. Bioinf. Bioeng, 1,443-450.
  2. Koppikar , SJ. et al. (2010) Aqueous cinnamon extract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human cervical cancer cell line (SiHa) through loss of mitochondrial membrane potential. BMC Cancer 10: 210. doi: 10.1186/1471-2407-10-210
  3. Singh, R. et al. (2009) Comparative analysis of cytotoxic effect of aqueous cinnamon extract from Cinnamomum zeylanicum bark with commercial cinnamaldehyde on various cell lines. Pharmaceutical biology, 47(12), 1174-1179.

 

Contact:

Ruchika fig2

Ruchika Kaul-Ghanekar, PhD

Sr. Associate Professor and Head

Cell and Translational Research Division

Interactive Research School for Health Affairs (IRSHA),Bharati Vidyapeeth University, Medical College Campus, Pune-Satara Road, Pune, Maharashtra, India

ruchika.kaulghanekar@gmail.com ; kaul_r@yahoo.com
SchemeFig 1. A scheme depicting the proposed mechanism of growth regulation of cervical cancer cells by Ficus religiosa aqueous extract (FRaq)

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