Abstract 5120: Cryptolepine a plant alkaloid, inhibits the growth of nonmelanoma skin cancer cells through inhibition of topoisomerase and induction of DNA damage

Topoisomerases are highly specialized nuclear enzymes that remove superhelical tension on chromosomal DNA that allows replication and transcription of DNA. Many cancer chemotherapeutic drugs used in the clinics inhibit tumor growth by targeting topoisomerase functions resulting in DNA damage and cancer cell death. Cryptolepine, a major alkaloid isolated from Cryptolepis sanguinolenta plant’s roots, has shown anti-malarial, anti-bacterial, anti-fungal, and anti-inflammatory activities. In the present study, we examined the therapeutic effect of cryptolepine on non-melanoma skin cancer cells (NMSCC), SCC-13 and A431 as an in vitro model, and underlying mechanism of action with special emphasis on topoisomerases and DNA damage check points. Western blot analysis and enzyme activity evaluation assays demonstrated that SCC-13 and A431 cells express comparatively higher levels of topoisomerases and higher enzymatic activities compared with normal human epidermal keratinocytes (NHEK). Topoisomerase expression and activity was greatly reduced after 24 h treatment with 2.5, 5.0 and 7.5 µM cryptolepine. Inhibition of topoisomerases expression and function by cryptolepine resulted in significant DNA damage and enhanced expression of DNA-PK expressions. Cryptolepine induced DNA damage activated DNA damage response proteins as demonstrated by increased phosphorylation of ATM/ATR, BRCA1, Chk1/Chk2 and γH2AX. Consequences of cryptolepine induced DNA damage were also associated with activation of p53 signaling cascade. Cryptolepine induced post-translational modifications such as phosphorylation and acetylation of p53, and inhibition of mdm2 protein expression resulted in activation and accumulation of p53 in SCC-13 and A431 cells. In turn, activated p53 enhanced protein expression of cyclin-dependent kinase inhibitors p16 and p21. Activation of p16 and p21 proteins downregulated expression of cyclin-dependent kinase 2 (CDK2), cyclin A, cyclin D1 and cyclin E and thus induced S-phase cell cycle arrest in NMSC cells with reduced expression of cell division proteins Cdc25a and Cdc25b. Cryptolepine treatment also enhanced release of cytochrome c from mitochondria due to increased disruption of mitochondrial membrane potential. Inhibition of topoisomerase activity and activation of DNA damage response signaling culminated in significantly reduced cell viability and enhanced cell death of SCC-13 and A431 cells. However, NHEK cells were least sensitive to cryptolepine induced cell death. Results of our study strongly suggest for detailed preclinical investigations in animal models to further assess its anti-skin cancer potential and its possible use in human patients. Note: This abstract was not presented at the meeting. Citation Format: Harish C. Pal, Santosh K. Katiyar. Cryptolepine a plant alkaloid, inhibits the growth of nonmelanoma skin cancer cells through inhibition of topoisomerase and induction of DNA damage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5120. doi:10.1158/1538-7445.AM2017-5120