Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells

Abstract

Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer.

Fig 1

Percentage cell viability of HT-29 cells treated with (A) Different crude extract from the rhizome of Alpinia mutica at different concentrations (μg/mL) for 72 hrs (B) FKB and APN at different concentrations (μM) for 72 hrs (C) HepaRG cells treated with FKB and APN at different concentrations ((μM) for 72 hrs (D) Vero cells treated with FKB and APN at different concentrations (μM) for 72 hrs. MTT assay was used to determine the IC₅₀ of the tested crude extract and phytocompounds.

Fig 2

(A) Expression of UCK2 mRNA in HT-29 cells analysed in 1% agarose gel. (a)Levels of UCK2 mRNA expression in cells treated with increasing concentration of crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts; (b) Levels of UCK2 mRNA expressed in cells treated with FKB at 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL); (c) Levels of UCK2 mRNA expressed in cells treated with APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). The housekeeping gene, GAPDH was used as loading control. C, Untreated control; D, DMSO used as negative control at a final concentration of 0.1%. (B) Western blot analysis of UCK2 protein expressed in HT-29 cells. (a) Levels of UCK2 protein expression in cells treated with increasing concentration of crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts. (b) Levels of UCK2 protein expressed in cells treated with FKB at a concentration of 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL). (c) Levels of UCK2 protein expressed in cells treated with APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). (C) Levels of UCK2 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT-29 cells treated with (a) Crude hexane and chloroform extract, and (b) Bioactive compounds of FKB and APN;. DC: DMSO treated control at a final concentration of 0.1%. Data are expressed as Mean±SD; ns: non-significant; *p<0.05; ns: non-significant compared to the DMSO control.

Fig 3

(A) Live cell imaging of the controls of HT-29 cells with SmartFlare Cy5-Uptake, Cy5-Scramble or Cy5-18S probes, imaged using fluorescence microscope in exposure settings at 20× magnification. (a) Uptake probe used as positive control that quenched on inside the cell. (b) Scramble probe used as negative control where the probe is not able to recognize any RNA sequence inside the cell nucleus. (c) Cells with 18S probe after 48 h of 0.1% (v/v) DMSO treatment as negative control. (B) Live cell imaging of HT-29 cells with SmartFlare Cy5-18S probes, and imaged using fluorescence microscope in exposure settings at 20× magnification. Cells with 18S probe after 48h treatment with crude extract of (d) hexane extract (21.05 μg/mL), (e) chloroform extract (19.09 μg/mL), (f) FKB (8.47 μg/mL), and (g) APN (13.12 μg/mL).

Fig 4. Levels of MDM2 and p53 proteins expressed in HT-29 cells.

(A) Level of proteins in cells treated with (a) crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts. (b) 25 μM (7.1 μg/ mL) of FKB at different time interval. (c) 25 μM (6.75 μg/mL) of APN at different time interval. (B) Level of MDM2 and p53 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT 29 cells treated with (a) Hexane and chloroform extracts (b) FKB, and (c) APN. Data are expressed as Mean±SD; ns: non-significant; *p<0.05; **p<0.01; ***p<0.01; ns: non-significant compared to the DMSO control. DC: DMSO used as negative control at a final concentration of 0.1%.

Fig 5. Levels of protein expression involved in mitochondrial apoptotic signalling pathway in HT-29 cells after 72 h incubation.

(A) Levels of protein expressed in cells treated with (a) crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts. (b) FKB at a concentration of 12.5 (3.55 μg/mL), 25 (7.1 μg/mL), and 50 μM (14.2 μg/mL) and (c) APN at a concentration of 12.5 (3.37 μg/mL), 25 (6.75 μg/mL), and 50 μM (13.5 μg/mL). (B) Levels of protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT-29 cells treated with (a) Hexane and chloroform extracts (b) FKB, and (c) APN. Data are expressed as Mean±SD; ns: non-significant; *p<0.05; **p<0.01; ***p<0.001 ns: non-significant compared to the DMSO control. DC: DMSO treated control at a final concentration of 0.1%.

Fig 6. Cell cycle analysis examined using flow cytometry on HT-29 cells after 72 h treatment.

(A) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) FKB at a concentration of 12.5 (3.55 μg/mL), (c) 25 (7.1 μg/mL), (d) 50 μM (14.2 μg/mL), and (e) Percentage of cell cycle distribution in different phases. (B) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) APN at 12.5 (3.37 μg/mL), (c) 25 (6.75 μg/mL), (d) 50 μM (13.5 μg/mL) concentrations, and (e) Percentage of cell cycle distribution in different phases. G0/G1, G2+M, and S are cell phases, respectively; subG0/G1 refers to cell death due to DNA fragmentation. Data are expressed as Mean±SD of three independent experiments, *p<0.001, ns: non-significant compared to the normal control.

Fig 7. DNA fragmentation analysed in 1% agarose gel after 72 h incubation with different concentration of either FKB or APN.

MW: DNA marker; DC: DMSO treated control; all concentrations are in μM.