Study on the Anticancer Activity of Prodigiosin from Variants of Serratia Marcescens QBN VTCC 910026

Abstract

Prodigiosin (Pg), a secondary metabolism produced by numerous bacterial species, is known as anticancer, antibacterial, antifungal, immunosuppressant, antioxidant, antimalarial properties. Pg has been tested for antitumor activity in many different cancer cell lines but studies in LU-1, KB cell lines, and tumor-bearing mice are still limited. In this study, Serratia marcescens QBN VTCC 910026 strain (GenBank: KX674054.1) was mutated using Ethyl Methanesulfonate (EMS) to increase the production of Pg. One strain known as EMS 5 was capable of increasing prodigiosin biosynthetic yield by 52% when compared to the wild-type strain. Red bacterial pigmented colonies containing Pg were collected from solid media, lysed with acetone, purified with toluene: ethyl acetate at a ratio of 9: 1 (v/v), and then used to evaluate the potential anticancer activity. The purity of Pg was confirmed using a high-performance liquid chromatography (HPLC) method which indicated a 98% rate. Pg chemical formula which was determined using ¹H-NMR and ¹³C-NMR spectroscopy, confirmed as prodigiosin (Pg). Human breast cancer cell lines MCF-7, oropharyngeal cancer KB, and particularly lung cancer LU-1 in vitro were used to test the anticancer activity of purified Pg compound. It showed a strong inhibitory ability in all the cancer cell lines. Furthermore, the isolated Pg had capable of inhibiting tumor growth, the tumor volume decreased by 36.82%, after 28 days. The results indicated that the bacterial prodigiosin from variants Serratia marcescens QBN VTCC 910026 strain is an encouraging fragment suitable for therapeutic applications.

Figure 1

Purification of Prodigiosin. (a) TLC chromatography of the purified prodigiosin passing through the silica-gel column; lane 1: standard Pg, lane 2, 3: purified Pg. (b) HPLC of purified prodigiosin.

Figure 2

Spectra of purified compound. (a). ¹H NMR proton spectrum. (b). ¹³C NMR spectrum of active prodigiosin purified from S. marcescens EMS 5.

Figure 3

Chemical structure of purified prodigiosin.

Figure 4

Inhibitory concentration of Prodigiosin (0.5; 1; 2; 4; 6; 8 and 10 μg/ml) on different cancer cell line KB, SK-LU-1, MCF7, H460 and HEPG2.

Figure 5

The effect of Pg on tumor in the thigh of mice before and after treatment. (a). The effect of Pg on tumor growth of mice after 28 days. Control: Healthy mice. Capecitabine: Mice were orally administrated with capecitabine at the dose of 200 mg/kg/day. PG: Mice were received with Pg at the dose of 2 mg/kg/day. Significance was determined using an unpaired two tailed t test: ∗∗∗∗p <0.0001, ∗∗∗p <0.001, ∗∗p <0.005, ∗p <0.05. Ns: Non-significant. Each value is expressed as mean ± SD. n = 10 per group. The average of tumor weight of all mice. (b). Tumor from mice without drug treatment. (c). Tumor was from mice orally administrated with capecitabine at the dose of 200 mg/kg. (d). Tumor was from mice injecting with Pg at the dose of 1 mg/kg (e).