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BY 4.0 license Open Access Published by De Gruyter Open Access May 24, 2023

Assessment of cytotoxic and apoptotic activities of the Cassia angustifolia aqueous extract against SW480 colon cancer

  • Maha Abdullah Momenah ORCID logo EMAIL logo , Alaa Ali Alqahtani , Fatima Abdullah AL Qassim , Amani Mohammed Alotaibi , Gadah Albasher and Wedad Saeed Al-Qahtani
From the journal Open Chemistry

Abstract

The current natural extract modalities for colorectal cancer are limited. This research seeks to assess the process of extracting the plant’s bioactive constituents from Cassia angustifolia and to show the anticancer role played by the plant’s aqueous extract at 0°C by identifying the genes that alter in expression after the Cassia angustifolia treatment in colon cancer cells. The bioactive components of Cassia angustifolia extract were revealed using gas chromatography-mass spectrometry analysis. The colon carcinoma cell lines (SW480) were treated with Cassia angustifolia macrophages at concentrations of 50, 150, and 200 µg/mL for 48 h. Apoptosis was examined by fluorescence-activated cell sorting analysis of Cassia angustifolia-treated and -untreated cells. Microarray analysis was performed by using human microarray chips (HG-U95A) for untreated and treated SW480 cells. Microarray data were confirmed by the reverse transcription polymerase chain reaction. The findings showed that the Cassia angustifolia aqueous extract at 0°C/24 h contained the entire absolute phenolic content of 28.43 mg/g and the entire absolute flavonoid content of 9.16 mg/g. Cassia angustifolia enhanced the hindrance of cell development and apoptosis and decreased glucose uptake. Western blot analysis showed induction in the expression of cleaved caspases 3 and 9 in SW480 cells. Microarray data identified 11 genes and 7 expressed sequence tags (ESTs) markedly altered in treated vs non-treated SW480 cells. Several of these genes have been embroiled in multiple malignancies. In conclusion, the current study provides remarkable new data for Cassia angustifolia extracted at 0°C/24 h. We discovered 7 ESTs and 11 genes that are significantly altered in the progression of SW480 cells treated with Cassia angustifolia therapy versus no treatment, with anticancer and apoptosis inducer effects.

1 Introduction

Colorectal cancer (CRC) has been ranked as the third most highly prevalent malignancy diagnosed worldwide as well as the fourth most common cause of death related to cancer worldwide. By 2030, the burden of the disease is projected to rise by 60% [1,2,3]. CRC, a multifactorial illness, involves lifestyle, environmental, and genetic risk factors [4]. Although it is highly affected by hereditary factors, most cases of CRC are sporadic developing over years through a stepwise way referred to as adenoma–carcinoma sequence [5]. Despite the great advancements in the control and treatment of cancer spread, significant opportunities for improvement and deficiencies remain. Several unwanted side effects appear at times during chemotherapy [6]. The negative side effects may be reduced by natural therapies, for example, the utilization of plant-extracted products for cancer treatment. There are several natural products that include dietary compounds originating from plants, vegetables, herbs, and spices, which have been utilized in cancer treatment for years because of their low toxicity, general availability, and safety [7,8].

The ancient medicinal plant Cassia acutifolia belongs to the Caesalpiniaceae family. It is normally referred to as Cassia Senna or Senna Makkai. Cassia angustifolia’s native countries are Yemen, Egypt, and Saudi Arabia [6]. The shrub grows rapidly from 5 to 8 m tall, highly cultivated for its leaves and fruits in the hot arid parts of Saudi Arabia. Antioxidant herbs and natural anticancer aid in managing the formation of activated oxygen species and free radicals or they can hinder their biological structures’ reaction [8,9].

The antioxidants comprise antioxidative enzymes like glutathione peroxidase, catalase, and superoxide dismutase as well as small molecules of nonenzymatic antioxidants, such as vitamin E, vitamin C, and glutathione [10,11,12,13]. Antioxidants are utilized to neutralize free radicals’ effects. Therefore, they shield humans from degenerative diseases and infection. Antioxidants are categorized as either synthetic or natural. Synthetic antioxidants are made up of gallic acid esters, tertiary butylated hydroquinone, butylated hydroxyl toluene, and butylated hydroxy anisole. These antioxidants efficaciously inhibit oxidation; they can serve as chelating agents like ethylene diamine tetra acetic acid (EDTA) that may bind metals minimizing their contribution to the process [10]. Nevertheless, antioxidants are suspected to lead to or enhance negative health impacts such as carcinogenesis and mutagenesis in humans [14,15]. Thus, synthetic antioxidants are being replaced by naturally arising antioxidants, which can limit free radical-linked diseases [16,17]. Globally, Cassia angustifolia’s consumption as a medicine against several diseases, and the studies published in the literature require further research in order to find the compounds causing its bioactivities.

Microarray analysis has emerged as a key technique of studying many genes in one experiment simultaneously on GeneChip oligonucleotide arrays (Affymetrix, Inc., Santa Clara, CA); a particular gene is denoted by 15–20 various 25-mer oligonucleotides, which function as idiosyncratic, sequence–sequence indicators. Additionally, a control element on the arrays is utilized as the mismatch sequence. Such probes are created to complement the sequence of reference with the exclusion of a homomeric base inconsistency on the central part. The presence of mismatched oligonucleotide permits cross-hybridization while the local background can be estimated and removed from the perfect match indicator. In GeneChip expression assay, the conversion of eukaryotic messenger RNA or messenger ribonucleic acid (mRNA) into biotinylated complementary deoxyribonucleic acid (DNA) or complementary deoxyribonucleic acid (cDNA), originally oligo-dT-primed cDNA, occurs [18]. Every sample undergoes hybridization into a different array. The levels of the transcript are computed through reference to the cDNA spikes that have known concentrations combined with the hybridization mixture. Variations in the levels of mRNA among samples are obtained by comparing two patterns of hybridization recorded on separate arrays for similar array types. For this research, the hydro extract is obtained at low temperatures (0°C) and recognized as an ancient medicinal plant utilized in the Kingdom of Saudi Arabia. Cassia acutifolia’s aqueous extract was evaluated because of its anticancer and antioxidant activities by particular experiments on genes’ set-in colon cell lines for cancer and the way they would potentially be utilized therapeutically against the proliferation and growth of colon cancer. They were additionally exposed to phytochemical filtering in order to assess the availability of bioactive compounds and secondary metabolites by the hydro extraction technique of Cassia acutifolia at low temperature (0°C). This study aims to evaluate the procedure of extracting bioactive ingredients at 50, 150, and 200 g/mL Cassia angustifolia for 48 h at low temperatures (0°C), and to demonstrate the anticancer role of the plant’s aqueous extract at 0°C by identifying the genes whose expression changes following Cassia angustifolia therapy in colon carcinoma cell lines (SW480) by using gas chromatography.

2 Materials and methods

2.1 Preparation of the aqueous plant extract

The leaves were obtained from Southern Saudi Arabia and were cleaned under running water, squashed, and blended with deionized water (1:20 w/v, Figure 1). For the preparation, 100 mL of water and 60 g of Cassia angustifolia leaves were used. Prior to the start of extraction, the mixture was kept below 0℃ for 24 h. The rough extract was centrifuged at 3,000 rpm for 15 min before lyophilizing the extract using a vacuum freezer-dryer (Model FDF 0350, Korea). The resultant extract yield obtained was 46.3% for every 100 g. Later, the concentrate was stored at 4°C as stock.

Figure 1 
                  The Cassia angustifolia leaves used in the present research, Saudi Arabia.
Figure 1

The Cassia angustifolia leaves used in the present research, Saudi Arabia.

2.2 Determination of total phenolic contents

The total phenolic aqueous content and the Cassia angustifolia organic extract content were assessed using the Folin–Ciocalteu technique [19].

2.3 Determination of total flavonoid contents

The aluminum chloride colorimetric technique was used to determine the total contents of flavonoids in the Cassia angustifolia extract [20].

2.4 Cell culture

The SW480 colon cancer cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and it was cultured in a medium supplemented with glutamine, penicillin, streptomycin, and fetal bovine serum. The medium was kept at a humidified temperature of 37°C in 5% carbon dioxide incubators [21,22].

2.5 Cell treatment

From a humidified hatchery, cells were seeded at 1 × 105 or 1 × 106 cells/well in 96 and 25 tissue-culture plates. An aqueous concentrate from Cassia angustifolia was then put into the culture media while the cells were treated with three different concentrations of 200, 150, and 50 µg/mL for 48 h.

2.6 Propidium iodide (PI)-based flow cytometric DNA fragmentation assay

Apoptosis was estimated using PI DNA staining. In brief, 2 × 106 adhering cells were broken by trypsinization. The floating dead and detached cells were centrifuged and washed two times with 1 mL of cold 1× phosphate-buffered saline (PBS) (Life Technologies). Then, the supernatant was extracted with 1 mL of cold 75% ethanol, and the cells were incubated at −20°C and spanned for 1 h. Afterward, these cells were cleaned with 1 mL of 1× PBS two times. After the final wash, 100 µL of the PI solution (50 µg/mL PI + 0.05 mg/mL RNase A; Sigma, St. Louis, MO, USA) was added. The cells were incubated for 2 h with protection from light before analysis at room temperature. DNA analysis was conducted by using fluorescence channel 3 in an Epic XL flow cytometer (Coulter Electronics, Inc., Miami, FL, USA) as indicated in the study of Huerta et al. [23]. For analysis of apoptosis, Cassia angustifolia-treated SW480 cells at concentrations of 200, 150, and 50 µg/mL for 2 days (48 h) were used.

2.7 Glucose uptake measurement

SW480 cells were treated with 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-d-glucose (2-NBDG) for 30 min post-Cassia angustifolia extract treatment at concentrations of 200, 150, and 50 µg/mL for 2 days (48 h). A flow cytometer (Becton-Dickinson, San Jose, CA) was used to determine the glucose uptake by SW480 cells.

2.8 RNA isolation and quantitation

The mRNA sample was obtained using the Qiagen an RNeasy Plus Mini Kit. The purity and concentration of RNA samples were determined using a Nano Drop® ND-1000 Spectrophotometer (Thermo Fisher Scientific). The integrity of the RNA sample was checked using an Agilent 2100 bioanalyzer (Agilent Technologies, Inc.) All RNA samples recorded 9.5 or greater integrity numbers, showing that the RNA samples did not degrade. There were technical triplicates for every concentration in the SW480 cells.

2.9 cDNA synthesis

About 5 µg RNA samples were reverse-transcribed into cDNA using a Superscript Choice System (Gibco-BRL, Rockville, MD, USA) and a T7-dT7 oligomer (Genset Inc., La Jolla, CA, USA). cDNA (double-stranded) was purified using phenol/chloroform production by Phase-Lock Gel (Fisher Scientific, Pittsburgh, PA, USA). cDNA (biotin-labeled) was manufactured in vitro using an RNA Transcription Labeling Kit (Enzo BioArray; Affymetrix Inc., San Jose, CA, USA). The synthesized cDNA was purified using an affinity resin column (Qiagen Inc., Valencia, CA, USA). About 10–15 µg of cDNA was fragmented by incubating at 86°C for 30 min in 40 mM Tris-acetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate.

2.10 Affymetrix analysis

Microarray analysis was performed by using human microarray chips (HG-U95A) (Affymetrix Inc., San Jose, CA, USA). Human Genome U95 Set has the most extensive transcript coverage for the human genome. The set examines the level of expression for >60,000 expressed sequence tags (ESTs) and human genes. U95A array has all complete-length genes. This array constitutes ∼12,000 sequences, which have already been characterized by disease association or function [18]. About 5 mg of cDNA, 0.1 mg/mL sonicated herring sperm DNA, and 1 mg of cDNA control cocktail were mixed with the hybridization buffer containing 0.5 mg/mL acetylated BSA, 0.01% Tween-20, 20 mM EDTA, 100 mM morpholine ethane sulfonic acid (MES), and 1 M sodium (Na). The hybridization mixture was exposed to 99°C heat for 5 min and then incubated at 45°C for 16 h, mixing in a rotisserie at 60 revs per minute. Hybridization was followed by the removal of the solution, and staining and automated washing were conducted using a rigorous buffer (0.1 M (Na), 0.01% Tween-20, 100 mM MES), a non-rigorous buffer (0.01% Tween-20, 6 SSPE, 0.005% Antifoam), antibody solution (3 µg/mL acetylated BSA, 0.05% Tween-20, 1 M Na, 100 mM MES, 0.005% antifoam), and streptavidin, R-phycoerythrin conjugate (SAPE) stain solution (streptavidin phycoerythrin) (10 µg/mL SAPE, 100 mM MES, 2 µg/µL acetylated bovine serum albumin [BSA], 0.05% Tween-20, 1 M Na, 0.005% antifoam). After staining and washing cycles, the array (probe) was scanned two times at a resolution of 3 µm using an HP Gene Array scanner (Hewlett-Packard, Palo Alto, CA, USA). Gene Chip Eukaryotic was used as the Hybridization Control Kit in estimating RNA’s abundance in samples.

2.11 Reverse transcription polymerase chain reaction (RT-PCR) analysis

The genes obtained from the analysis of microarray were searched with accession numbers in the GenBank’s Entry database, while primers were created from the cDNA sequence in every particular gene, as shown in Table 1. Then, the RT-PCR process was conducted as previously explained. In brief, the cell line (SW480) was treated or untreated with Cassia angustifolia at concentrations of 200 and 150 µg/mL for 2 days (48 h), and cumulative RNA was obtained from cell pellets (frozen) using an Isolation Kit (Qiagen RNeasy). Random hexamers were used to prime first-strand cDNA (Gibco-BRL, Rockville, MD, USA). cDNA’s synthesis was done with the Superscript Choice System from Gibco-BRL (Rockville, MD, USA). In order to magnify the different PCR products, primers were used as shown in Table 1. There was amplification of the positive control by PCR utilizing primers, particularly for the housekeeping gene: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: 5′-TTT GGT ATT GAA GAG GGC CT-3′ and reverse: 5′-ATT AAA GCC AAA GTA AAA GC-3′. The RT-PCR negative control was included by conducting parallel reactions in every cDNA with control primers (GAPDH) and the exception of the reverse transcriptase enzyme. The PCR conditions were as follows: 94°C heat for 30 s, 52–55°C heat for 30 s, and 72°C heat for 30 s in 30 cycles. The products from PCR were separated using an agarose gel (2%) containing ethidium bromide and then visualized on UV light. The intensity of all bands was examined using an image analyzer (Bio-Rad, Burlington, MA, USA), and the intensity for all experimental primers of 200 and 150 µg/mL Cassia angustifolia-treated SW480 cell line was collated to that of untreated cells.

Table 1

Real-time primer sequences for PCRs

Gene symbol Gene name Primer sequence
MCT Monocarboxylate transporter Forward: 5′-TTC ACT ATC GGC TTC AGC AA-3′
Reverse: 5′-CCG ATG CCA CTC ATG GAC AC-3′
GAD67 Human glutamate decarboxylase Forward: 5′-CTA GGG GCC AAG GGA AAT GC-3′
Reverse: 5′-CCT CCC ACC ACC AAG GTC CAG-3′
P19 Human serine proteinase inhibitor Forward: 5′-TGT GTC CCC AGA TCC CCA CT-3′
Reverse: 5′-AAA GCA CAG GGT CGC CAG AG-3′
GSTM3 Human glutathione transferase M3 Forward: 5′-ACA TCG CTC GCA AGC ACA AC-3′
Reverse: 5′-ATC CAG GCA CTT GGG GTC AA-3′
TRAM1 Translocation-associated membrane protein 1 Forward: 5′-ACA GGA TTG TCG GGG AAG GAG-3′
Reverse: 5′-TGA AGC CCA CAT CAG CAG TCA-3′
Cyclin D1 Human cyclin D1 Forward: 5′-GCA GTA GCA GCG AGC AGC AG-3′
Reverse: 3′-CTA CAC CGA CGG CTC CAT CC-3′
ATM Human ataxia-telangiectasia locus protein gene Forward: 5′-TGC AGG CAC ATG CTA CCA CA-3′
Reverse: 5′-TGC CCC GAT TCA ATC TCA CA-3′
TFAR15 Homosapiens apoptosis-linked protein Forward: 5′-CGA CCA GAG CCA GAA TTC CAA-3′
Reverse: 5′-TGC CCT GCG GTT CTG GTA TT-3′
CO-029 Human CO-029 Forward: 5′-GCA GGT GGC GAC AGG TAT CC-3′
Reverse: 5′-TCC CCA ATC AGC AGC TCC AT-3′
CDC20 Cell division cycle 20 Forward: 5′-ATTCCCAGGTGTGCTCCATC-3′
Reverse: 5′-GCCATGGTTGGGTACTTCCA-3′
ESA Human surface antigen (ESA) mRNA Forward: 5′-TGG TTT CAG GGG GCT GTT GT-3′
Reverse: 5′-TCG GCC GTC TCT ACG TCC TC-3′

2.12 Microarray data acquisition and analysis

After scanning the microarray, images were loaded into Dchip software [24]. The software package calculates two summary rates for every gene. One is a model-based expressing value that encapsulates the approximate level of expression on 20 separate probes, and the other is the absent/present call. The call is a decision regulation utilizing several details in the pairs of probes to determine whether there was any mRNA correlating with that specific gene composing the sample. Additionally, genes were regarded as having substantial differential expression in case they attain the following criteria: (i) a fold change higher than 2 on one comparison and (ii) a substantial main impact in the ANOVA’s model. The ANOVA’s coefficient P-values were based on two-sided tests that were unadjusted for several comparisons. Thresholds were considered substantial at P ≤ 0.05 for every effect (i.e. cell treatment and type).

2.13 Western blot analysis

Colon carcinoma cell lines were incubated with or without 200 and 150 µg/mL Cassia angustifolia for 48 h. Then, the cells were detached by trypsin treatment and lysed at 4°C in a radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 50 mM Tris-HCl [pH 7.4], 150 mM NaCl, and 0.25% sodium deoxycholate, supplemented with a protease self-regulator cocktail, Complete Mini’s tablet from Roche, Indianapolis, IN, USA). Subsequently, 40 µg cell lysates were electrophoresed with 15% SDS-PAGE (Bio-Rad, Hercules, CA, USA) and subjected to Western blot analysis. In addition, proteins were passed on onto Hybond nitrocellulose membranes (Amersham, Arlington Heights, IL, USA) from gels in semi-dry transblotting cells (Bio-Rad, Hercules, CA, USA). The membranes were obstructed with 5% skim non-fat milk/PBS at room temperature for 1 h, followed by incubation with an appropriate antibody for 1 h at room temperature. Rabbit anti-caspase-3 polyclonal antibody was from mice while anti-caspase-9 polyclonal antibody was purchased from Cell Signalling (New England Biolabs, Beverly, MA, USA). After cleaning with PBS/0.1% Tween-20 twice, the membranes were incubated with anti-rabbit IgG Ab or horseradish peroxidase-conjugated anti-mouse (New England Biolabs, Beverly, MA, USA) for 30 min. After cleaning with Tris-buffered saline/0.1% Tween-20 thrice, the membranes were developed with the LumigloWestern blot detection kit (New England Biolabs, Beverly, MA, USA).

2.14 Analysis of statistics

Analytical examinations were done using SigmaStat programming adaptation 3.5 (Systat Software, San Jose, CA, USA). The quantitative results were expressed in terms of mean ± standard deviation. P < 0.05 values were considered statistically significant.

3 Results

3.1 Phenolic and flavonoid contents

Absolute phenolic and flavonoid contents of the Cassia angustifolia aqueous concentrate are shown in Table 2. The absolute flavonoid content and absolute phenolic content of Cassia angustifolia were 9.16 ± 1.68 and 28.43 ± 1.29 mg/g, respectively.

Table 2

Total phenolic and flavonoid contents in the Cassia angustifolia profile of watery extract (n = 10)

Test Concentration (mg/g)
Total phenolic content (TPC) 28.43 ± 1.29
Total flavonoid content (TFC) 9.16 ± 1.68

The values are an average of three replicates ± Standard Deviation: mg GA/g FW – milligram gallic acid equivalents per gram fresh weight of tissue; QE/g FW – milligram quercetin equivalents per gram fresh weight of tissue.

3.2 Apoptosis induction with Cassia angustifolia

To assess the Cassia angustifolia’s impact on colon carcinoma cells, colon cells were treated or untreated with different concentrations (200, 150, and 50 µg/mL) of Cassia angustifolia for 48 h. The SW480 cells treated with 50 µg/mL indicated a lesser portion of cells encountering apoptosis in comparison to those treated with 200 and 150 µg/mL (P ≤ 0.05). Figure 2 shows that Cassia angustifolia treatment led to a concentration-dependent apoptosis induction at two concentrations (200 and 150 µg/mL). Nevertheless, the biggest difference was recorded for 200 µg/mL because it had differential apoptosis degrees relative to the untreated cells (P ≤ 0.001). These results show that the SW480 cells, i.e. non-metastatic cell lines were more apoptosis induced for concentrations of 200 and 150 µg/mL Cassia angustifolia treatment in comparison to the concentration of 50 µg/mL as well as untreated cells (P ≤ 0.01).

Figure 2 
                  The impact of Cassia angustifolia treatment on apoptosis induction in SW480 cells. Then, apoptosis was determined using PI staining on triplicate samples. Treatment with Cassia angustifolia resulted in concentration-dependent apoptosis induction. Nevertheless, the apoptosis induction in the SW480 cells was substantial (P ≤ 0.001) at higher doses of 200 and 150 µg/mL Cassia angustifolia treatments for 48 h in comparison to the untreated cells. Besides, apoptosis induction in the SW480 cells to the level of 50 µg/mL Cassia angustifolia treatment in a day was a bit higher (not substantial) in comparison to the untreated cells. The data are expressed in terms of means ± SEM. Variation in effects of treatment was significant at *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2

The impact of Cassia angustifolia treatment on apoptosis induction in SW480 cells. Then, apoptosis was determined using PI staining on triplicate samples. Treatment with Cassia angustifolia resulted in concentration-dependent apoptosis induction. Nevertheless, the apoptosis induction in the SW480 cells was substantial (P ≤ 0.001) at higher doses of 200 and 150 µg/mL Cassia angustifolia treatments for 48 h in comparison to the untreated cells. Besides, apoptosis induction in the SW480 cells to the level of 50 µg/mL Cassia angustifolia treatment in a day was a bit higher (not substantial) in comparison to the untreated cells. The data are expressed in terms of means ± SEM. Variation in effects of treatment was significant at *P < 0.05, **P < 0.01, ***P < 0.001.

3.3 Inhibition of glucose uptake by Cassia angustifolia

After we have shown that Cassia angustifolia led to cells’ apoptosis induction, we examined the impact of treatment on the uptake of glucose and mitochondrial activity in the SW480 cells. However, if the absorption of glucose was inhibited, a subsequent suppression of cell growth followed. We found out that glucose uptake (2-NBDG) was impacted by the aqueous extract of Cassia angustifolia. Glucose uptake restraint was made easier in a dose-subordinate way for SW480 cells treated with the aqueous concentrate of Cassia angustifolia for 2 days (48 h), as indicated in Figure 3.

Figure 3 
                  Glucose uptake restraints in the SW480 cells treated with the aqueous extract (Cassia angustifolia) inducted at higher doses of 200 and 150 µg/mL Cassia angustifolia treatment in 2 days (48 h). Although the inhibition of glucose uptake in the SW480 cells having 50 µg/mL in Cassia angustifolia treatment spanning 48 h was a bit higher (not substantial) in comparison to the untreated cells. The uptake of glucose was measured using flow cytometry. Furthermore, data are expressed in terms of mean ± SEM. The variation in treatment impact was regarded as significant (*P < 0.05, **P < 0.01, ***P < 0.001).
Figure 3

Glucose uptake restraints in the SW480 cells treated with the aqueous extract (Cassia angustifolia) inducted at higher doses of 200 and 150 µg/mL Cassia angustifolia treatment in 2 days (48 h). Although the inhibition of glucose uptake in the SW480 cells having 50 µg/mL in Cassia angustifolia treatment spanning 48 h was a bit higher (not substantial) in comparison to the untreated cells. The uptake of glucose was measured using flow cytometry. Furthermore, data are expressed in terms of mean ± SEM. The variation in treatment impact was regarded as significant (*P < 0.05, **P < 0.01, ***P < 0.001).

3.4 Activation of caspase cascade by Cassia angustifolia

The results indicated that Cassia angustifolia led to the inhibition of glucose uptake and apoptosis induction in the SW480-treated cells. Thus, the impact of Cassia angustifolia’s treatment on caspase cascade in SW480 cells was also evaluated. Cells were treated or untreated (control) with two doses of 200 and 150 µg/mL Cassia angustifolia for 2 days (48 h). Western blot analysis indicated that the SW480 cells show comparable amounts of caspases 9 and 3. Figure 4 shows that Cassia angustifolia treatment indicated induction in both caspases 3 and 9 in SW480 cells. Besides, the data showed that the sensitivity of SW480 cells toward Cassia angustifolia is equal to those of caspases 3 and 9.

Figure 4 
                  Impact of Cassia angustifolia treatment (200 and 150 µg/mLml) for 48 h on the expression of caspase proteins from Western blot analysis. The pro-caspases 9 and 3 expressed comparable values in SW480 cells for the two doses (200 and 150 µg/mL) with ß-actin. Comparably, activated caspases were also present in SW480 cells with the control after the treatment of Cassia angustifolia.
Figure 4

Impact of Cassia angustifolia treatment (200 and 150 µg/mLml) for 48 h on the expression of caspase proteins from Western blot analysis. The pro-caspases 9 and 3 expressed comparable values in SW480 cells for the two doses (200 and 150 µg/mL) with ß-actin. Comparably, activated caspases were also present in SW480 cells with the control after the treatment of Cassia angustifolia.

3.5 Microarray data

The set of data was composed of 12,625 genes. First, the genes that were lacking in the tested samples were removed and this stage gave 10,436 genes. Second, the development of the analysis of variance (ANOVA) model was entailed. Another ANOVA model catered for the other 2,189 genes, which attained the expression measure as the resultant estimate to determine the Cassia angustifolia’s effective concentrations in SW480 cells. Eighty-one genes were present with a substantial impact on the ANOVA model. Afterward, the fold-change between every experimental condition was computed. Fold changes of less than −2 and more than 2 were significant. Of 81 genes having substantial ANOVA impact, 43 had substantial fold-changes as shown in Table 3. From the 43 genes, 18 genes with expression changes had statistical significance relative to the Cassia angustifolia treatment (200 and 150 µg/mL), while only 2 of 18 genes were significantly expressed to the Cassia angustifolia treatment (50 µg/mL). In addition, 7 genes were ESTs while 11 were properly characterized genes, many of which were packed to genes entailed in the metabolism of cells.

Table 3

ANOVA analysis for genes’ differential expression noted from microarray analysis, which was substantial in SW480 filtered with Cassia angustifolia effects: 11 of 43 genes were significantly affected by Cassia angustifolia treatment (200,150, and 50 µg/mL)

Concentration (µg/mL) Significant ESTs Non-significant Total
50 2 7 34 43
150 18 7 25 43
200

Cassia angustifolia effects were considered markedly significant at P ≤ 0.05.

3.6 RT-PCR data

The microarray outcomes for all 11 genes were validated through RT-PCR. Following microarray results, all 11 genes did not indicate a variation in the expression of the gene relative to the Cassia angustifolia treatment (50 µg/mL). However, 9 of 11 genes showed different gene expressions through RT-PCR in response to the Cassia angustifolia treatment (150 and 200 µg/mL) (Table 4 and Figure 5).

Table 4

Genes showing a substantial change among the concentrations tested: 200, 150, and 50 µg/mL for 48 h with Cassia angustifolia in the SW480-treated cells in comparison to the untreated cells by RT-PCR analysis

Gene symbol Gene expression Fold-change (50 µg/mL) P-value Fold-change (150 µg/mL) P-value Fold-change (200 µg/mL) P-value
MCT Upregulated 0.9322 N.S. 2.25098 0.000 2.41044 0.000
GAD67 Downregulated −0.85011 N.S. −2.85507 0.000 −3.18451 0.000
P19 Upregulated 1.30188 N.S. 2.91732 0.000 3.62305 0.000
GSTM3 Downregulated −0.94301 N.S. −2.62413 0.000 −3.77489 0.000
TRAM1 Downregulated −0.90672 N.S. −2.5985 0.000 −3.40283 0.000
Cyclin D1 Downregulated −0.97289 N.S. −2.34251 0.000 −3.83403 0.000
ATM Upregulated 1.26216 N.S. 2.78734 0.000 3.20462 0.000
TFAR15 Upregulated 1.45301 N.S. 2.84536 0.000 3.32012 0.000
CO-029 Downregulated −1.04032 N.S. −3.79423 0.000 −4.31051 0.000
CDC20 Downregulated −1.15121 N.S. −3.02115 0.000 −3.94231 0.000
ESA Downregulated −1.01782 N.S. −2.89127 0.000 −3.52734 0.000
Figure 5 
                  Heat map of fold-change expression levels with the concentrations tested: 200, 150, and 50 µg/mL for 48 h of Cassia angustifolia in the SW480-treated cells in comparison to the untreated cells by RT-PCR analysis (n = 3). Rows represent fold-change expression levels for the selected genes and the columns represent concentrations (200, 150, and 50 µg/mL). Red blocks show high and green blocks low fold-change levels in comparison to the SW480-untreated cells.
Figure 5

Heat map of fold-change expression levels with the concentrations tested: 200, 150, and 50 µg/mL for 48 h of Cassia angustifolia in the SW480-treated cells in comparison to the untreated cells by RT-PCR analysis (n = 3). Rows represent fold-change expression levels for the selected genes and the columns represent concentrations (200, 150, and 50 µg/mL). Red blocks show high and green blocks low fold-change levels in comparison to the SW480-untreated cells.

4 Discussion

Medicinal plants produce biologically lively natural products because of their curative characteristics that have been examined for years [25,26,27 28]. This study aimed to evaluate the likely anticancer properties of the Cassia angustifolia sedimentary extract below 0°C. Among the extracts, the methanol extract of Cassia angustifolia indicated the highest interaction with a broad range of activity in case of cancer proliferation [29]. Flavonoids and phenolics are ancillary metabolites obtained from phenylalanine and tyrosine with potent antioxidant and antibacterial activities [30]. The results showed that isolated flavonoids of Cassia angustifolia have substantial antioxidant activities in oxidative stress [31,32,33,34]. From the aqueous extract, poor extraction of 2,2-diphenyl-1-picrylhydrazyl with 67.7% scavenging activity was obtained. This is because phenols and flavonoids causing antioxidant activity are poorly extracted into an aqueous extract [8,35,36]. In contrast, our results showed that at 0°C, the cold aqueous extract has greater quantities of phenolic and flavonoid compounds because of the low temperature of the watery solvent, which preserves phenols and flavonoid ingredients after extraction. However, no substantive anticancer research on Cassia angustifolia has been recorded earlier. A few studies showed that the ethanol and methanol extracts of Cassia angustifolia have anticancer properties [29,32,37]. Earlier, it was shown that auxiliaries such as flavonoids can result in anticancer activities [38]. This research supported the concept of anticancer activity due to the presence of the isolated flavonoids that were traced in the Cassia angustifolia aqueous extract although at 0°C. From earlier investigation, scutellarin (a bioactive compound) was obtained from Angustifolia; it had anticancer properties by substantially suppressing the HT1080 human fibrosarcoma cells’ proliferation through apoptosis induction. Similar research (in vivo experiment) indicated that the tumor’s weight and size were reduced post-scutellarin treatment [29,39]. Furthermore, extricate Cassia angustifolia systems enhanced the hindrance of cell development and apoptosis, and reduced glucose uptake. On the other hand, protein expressions for both caspases 3 and 9 became activated after Cassia angustifolia treatment. Caspases are important proteins that induce carcinogenesis. Caspases are either executor caspase or initiator caspase, i.e. caspase 9 (executor caspase) and caspase 3 (initiator caspase). Both the executor and initiator pro-caspases were enhanced in the SW480 cells after treatment with 200, and 150 µg/mL Cassia angustifolia for 48 h through Western blotting. Thus, the outcomes of the current research indicated that apoptosis induction in the SW480 colon cancer cell line after Cassia angustifolia treatment involves subsequent induction of caspase 9 and caspase 3 [40]. Therefore, we decided to conduct a concurrent analysis for a thousand genes after apoptosis induction with Cassia angustifolia to further validate the anticancer agent. In order to identify several genes in apoptosis in response to Cassia angustifolia, a microarray study was conducted in Cassia angustifolia treated and SW480 untreated cells.

About 12,625 genes are present on the oligonucleotide array. Approximately 18 genes were highly likely accountable for Cassia angustifolia-induced apoptosis on SW480 cells. From the 18 genes regulated differentially, 11 showed cDNA sequences on GenBank. Therefore, this facilitated primers’ design for RT-PCR that confirmed different expressions from microarray analysis in all 11 genes. There are better sensitive techniques to examine differential expression by these genes, for example, real-time PCR.

Our analysis showed that a substantial number of transcripts around the genes are involved in the metabolism of cells. Several genes have been indicated to be imperative during oncogenesis. For example, monocarboxylate transporter overexpression has been shown to occur in central nervous system malignancies [41] as well as P19, the inhibitor of novel serine proteinase, in squamous cell carcinoma for lungs [42]. Similarly, the activity of glutamic acid decarboxylase 67 (GAD67) and gamma-aminobutyric acid are high in colon and breast neoplastic tissues [30]. The activity of glutathione transferase has been noted to be low in control patients’ colonocytes versus the ones at high risk of contracting colon cancer [8,43]. Besides, overexpressed glutathione transferase causes resistance (chemotherapeutic) in the cell lines for ovarian cancer [44]. Furthermore, ATM mutations lead to the formation of intermittent human cancers like B-cell chronic lymphocytic leukemia and T-cell prolymphocytic leukemia [44,45]. Mutations of the human ataxia-telangiectasia locus protein gene (ATM) as a result of aberrant substitute splicing are identifiable for colon cancer [46,47]. Other genes recorded in the analysis involve genes taking part in the adhesion of cells. Adhesion between cells is an imperative process during metastasis. Previously, we have characterized some adhesion molecules on the system of cell lines, which are expressed while others are lost. In vivo, cell adhesion loss would facilitate the migration of aberrant cells in the metastasis process [18,48,49]. In this study, we found out that tetra-spanning colocalization of the tetraspanins (CO-029) was substantially altered. CO-029 has appeared overexpressed in hepatocellular carcinoma while it may be a crucial gene product enhancing tumor metastasis [50,51]. The epidermal surface antigen (ESA), an extracellular epidermal molecule, has been thought to contribute to epidermal intercellular adhesion and showed downregulation expression in the analysis. Also, the cell division cycle 20 (CDC20) molecule has been thought to contribute to cell division and was among the differentially expressed genes in the analysis. Besides, the analysis led to the discovery of seven ESTs substantially altered in our study. Unless the genes correlating with the ESTs were fully characterized, the importance of the EST outcomes in the Cassia angustifolia-induced apoptosis remains unclear.

5 Conclusion

In this research, we identified 7 ESTs and 11 genes that are substantially changed in the progression of SW480 cells with or without Cassia angustifolia treatment. Cassia angustifolia being a potent apoptosis inducer, these anticancer impacts have been linked to the high phenolic and flavonoid concentrations having higher effective anticancer in comparison to the alcoholic extracts and aqueous extract (boiled water as a solvent). Thus, it is clear that Cassia angustifolia remains a potential anticancer medication in vitro while further study in vivo may be conducted for formulation of a cost-effective Cassia angustifolia drug.

Acknowledgments

The authors would like to acknowledge Princess Nourah bint Abdulrahman University Researchers Supporting Project number (PNURSP2023R224).

  1. Funding information: This study was financially supported by Princess Nourah bint Abdulrahman University Researchers Supporting, Project number (PNURSP2023R224).

  2. Author contributions: M.A.M.: conceptualization, methodology, software data curation, visualization, investigation, writing–reviewing, editing, and submitted the paper as a corresponding author. A.A. Al-Q., F.A. AL-Q., and A.M. Al-O., methodology, investigation, writing–reviewing and editing. G.I. Al-B.: methodology, investigation, software data curation, writing–reviewing, and editing. W.S. Al-Q.: carried out the design of the study, conceptualization, methodology, performed the statistical analysis, software data curation, visualization, investigation, writing – original draft. All authors read and approved the final manuscript.

  3. Conflict of interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

  4. Ethical approval: The study followed the principles established by the Deanship of Scientific Research for Princess Nourah Bint Abdulrahman University. All human blood and urine samples, plant authentication, relevant data, and the research protocols were performed in accordance with the Institutional Review Board and approved by the ethical committee of KACST from Riyadh, Saudi Arabia (Protocol No. H-01-R059, IRB LOG number 20-0242). The study protocols complied with the requirements, relevant regulations, and guidelines of the declarations of Helsinki.

  5. Data availability statement: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

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Received: 2023-03-17
Revised: 2023-04-18
Accepted: 2023-04-30
Published Online: 2023-05-24

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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