Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines

doi:10.1016/j.rsci.2024.04.002

Abstract

Abstract:

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures (RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects.

Key words: anticancer, cytotoxicity, metabolite profiling, plant tissue culture, rice callus suspension culture, secondary metabolite

Anuradha Kumari, Wusirika Ramakrishna. Anticancer Activity of Rice Callus Suspension Cultures from Aromatic Varieties and Metabolites Regulated in Treated Cancer Cell Lines[J]. Rice Science, 2024, 31(4): 449-462.

Figures/Tables 9

Fig. 1. Rice calli grown on Murashige and Skoog medium under sterile conditions (A), and rice callus suspension cultures (RCSC) of Basmati 386 (B) and Basmati 1121 (C) showing a characteristic absorbance pattern in the ultraviolet range.

Fig. 2. Alamar Blue assay for A549 lung cancer cell line treated with different dilutions of Taxol, plant medium (PM), rice callus suspension culture (RCSC), and rice seed extract (SE) for 24 to 96 h. RCSC 386 and RCSC 1121 represent RCSC from aromatic rice varieties Basmati 386 and Basmati 1121, respectively, and SE 386 and SE 1121 represent SE from Basmati 386 and Basmati 1121, respectively. CK, PM, and Taxol represent untreated, negative, and positive controls, respectively. The means of cell viability rate relative to untreated control, along with standard deviation values, were presented. Student’s t-test was performed to assess the significant levels at P < 0.05 (*), P < 0.005 (**), and P < 0.001 (***).

Fig. 3. Alamar Blue assay for HT-29 colon cancer cell line treated with different dilutions of Taxol, plant medium (PM), rice callus suspension culture (RCSC), and rice seed extract (SE) for 24 to 96 h. RCSC 386 and RCSC 1121 represent RCSC from aromatic rice varieties Basmati 386 and Basmati 1121, respectively, and SE 386 and SE 1121 represent SE from Basmati 386 and Basmati 1121, respectively. CK, PM, and Taxol represent untreated, negative, and positive controls, respectively. The means of cell viability rate relative to untreated control, along with standard deviation values, were presented. Student’s t-test was performed to assess the significant levels at P < 0.05 (*), P < 0.005 (**), and P < 0.001 (***).

Fig. 4. Analysis of live/dead cells by Hoechst 33342 and propidium iodide (PI) staining, as visualized by a confocal laser scanning microscope after treatment with 1:5 dilution of plant medium (PM), RCSC 386, and RCSC 1121 for 72 h. A, A549 lung cancer cell line. B, HT-29 colon cancer cell line. RCSC 386 and RCSC 1121 represent rice callus suspension culture (RCSC) from aromatic rice varieties Basmati 386 and Basmati 1121, respectively. Yellow and blue arrows indicate cell shrinkage and nuclear fragmentation, respectively.

Fig. 5. Cell cycle analysis using flow cytometry for Annexin V and propidium iodide based assays. A and B, Lung cancer cell line A549 was stained with Annexin V (A) and propidium iodide (B). C and D, Colon cancer cell line HT-29 was stained with Annexin V (C) and propidium iodide (D). RCSC 386 and RCSC 1121 represent rice callus suspension culture (RCSC) from aromatic rice varieties Basmati 386 and Basmati 1121, respectively. The analysis was performed after treatment for 72 h. In A and C, the four quadrants, upper left (UL), upper right (UR), lower right (LR), and lower left (LL), represent necrotic, late apoptotic, early apoptotic, and living cells, respectively.

Fig. 6. Scratch assay displaying effect of rice callus suspension culture (RCSC) on cell migration. A, In lung cancer cell line A549. B, In colon cancer cell line HT-29. RCSC 386 and RCSC 1121 represent rice callus suspension culture (RCSC) from aromatic rice varieties Basmati 386 and Basmati 1121, respectively. Images were obtained at 24, 48, and 72 h after treatment. The gap size was determined using ImageJ software.

Fig. 7. Heat maps generated by hierarchical clustering analysis showing differentially expressed metabolites in RCSC 386 and RCSC 1121 treatment vs untreated control and plant medium (PM) in HT-29 cell line (A) and A549 cell line (B). Up-regulated and down-regulated metabolites are shown in red and blue colors, respectively. RCSC 386 and RCSC 1121 represent rice callus suspension culture (RCSC) from aromatic rice varieties Basmati 386 and Basmati 1121, respectively.

Fig. 8. Metabolites in HT-29 (A) and A549 (B) cell lines with maximal variable importance in prediction (VIP) scores identified by partial least squares discriminant analysis using MetaboAnalyst. Colored boxes indicate the relative concentrations of the metabolites in each group (PM, RCSC 1121, and RCSC 386), with up-regulation and down-regulation denoted by red and blue colors, respectively. RCSC 386 and RCSC 1121 represent rice callus suspension culture (RCSC) from aromatic rice varieties Basmati 386 and Basmati 1121, respectively. PM, Plant medium.

Table 1. Impact values and key metabolites of pathways analyzed by Metaboanalyst.

Cell line	Pathway name	P-value	Impact value	Key metabolite
HT-29	Glyoxylate and dicarboxylate metabolism	1.824 × 10^-4	0.106	l-Glutamic acid
HT-29	Glycine, serine and threonine metabolism	5.161 × 10^-5	0.246	l-Threonine
A549	Glycerolipid metabolism	2.396 × 10^-4	0.237	Glycerol
	Arginine biosynthesis	0.022	0.117	l-Glutamic acid
	Alanine, aspartate and glutamate metabolism	0.048	0.197	l-Glutamic acid
	Arginine and proline metabolism	0.048	0.164	l-Proline, l-Glutamic acid
	Tyrosine metabolism	0.048	0.140	l-Tyrosine
	d-Glutamine and d-glutamate metabolism	0.025	0.500	l-Glutamic acid
	Phenylalanine, tyrosine and tryptophan biosynthesis	0.013	0.500	l-Tyrosine

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