Antitumor Effects of L-citrulline on Hela Cervical Cancer Cell Lines

Abstract

Aim: Cervical cancer is the deadliest gynecological malignancy. This study aims to examine the anticancer effects of L-citrulline on HeLa cell culture.

Materials and methods: HeLa cells were cultured in complete Eagle's minimum essential medium. HeLa cells were seeded in 96-well plates and incubated with L-citrulline. After incubation, MTT dye was added and incubated. Annexin- V technique was used to test the apoptosis. The activated caspases of HeLa cells by L-citrulline exposure were measured with the Caspase 3/7 technique. One-way variance analysis was conducted for statistical analysis by using GraphPad Prism 6.0 for Windows.

Results: L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner in application times of 24 and 48 hours. The IC₅₀ dose of L-citrulline was 0.19 and 0.16 mg/mL at 24 and 48 hours, respectively. When HeLa cells were exposed to an IC₅₀ dose of L-citrulline for 24 hours, the percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.75%, 23.05%, and 12.75%, respectively. The differences in the wideness of the scratch area were observed at the initial stage and after 24 hours of applying L-citrulline.

Conclusion: L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner. Based on Annexin and Caspase findings, it can be concluded that L-citrulline exerted a pro-apoptotic effect on HeLa cells in only a short exposure time. L-citrulline also showed a migration inhibitory effect. The findings of this study indicate L-citrulline to be worthy of investigation for its anticancer activities in vitro and in vivo, and as a candidate for cancer therapy.

Keywords: Annexin-V; HeLa; L-citrulline; MTT; anticancer effects; migration assay.

Fig. (1)

Growth inhibition activity of L-citrulline on HeLa cells. IC₅₀ dose of L-citrulline 0.19 mg/mL for 24 hours (*: p<0.5).

Fig. (2)

Growth inhibition activity of L-citrulline on HeLa cells. IC₅₀ dose of L-citrulline 0.16 mg/mL for 48 hours (*: p<0.5).

Fig. (3)

Apoptosis profiles of HeLa cells. A) Control HeLa cells; Live cells 99.95% were detected. Percentages of late apoptotic, early apoptotic and dead cells were detected as 0.00%. B) HeLa cells were exposed to an IC50 dose of L-citrulline for 24 hours. 63.45% of cells were live. The percentages of the dead, early apoptotic and late apoptotic cells were detected to be 0.75%, 23.05% and 12.75%, respectively.

Fig. (4)

Caspase-dependent apoptosis profiles of HeLa cells. A) Untreated HeLa cells (95.90% live, 3.15% dead, 0.85 apoptotic/dead, and 0.10% apoptotic cells). B). HeLa cells treated with IC50 concentration of L-citrulline for 24 hours (91.80% live, 3.75% dead, 4.30% apoptotic/dead, and 0.15% apoptotic cells).

Fig. (5)

Migration capability of HeLa cells. A) Control HeLa cells at the 0th hour. B) Control HeLa cells after 24 hours. C) HeLa cells treated with L-citrulline at the 0^th hour. D). HeLa cells treated with L-citrulline at the 24th hour.

Fig. (6)

Migration percentage of HeLa cells. A) Control HeLa cells at the 0th hour. B) Control HeLa cells after 24 hours. C) HeLa cells treated with L-citrulline at 0^th hour. D) HeLa cells treated with L-citrulline at 24^th hour.