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Open Access | Published: 2022 - Issue 5

FLAVONOIDS OF CAPPARIS CARTILAGINEA FRUIT EXTRACT EFFECT ON WOUND HEALING IN HUMAN PROSTATE CANCER CELL LINE

Hala Salim Sonbol1*, Maram Maram Bakr Hosawi1

 

  1. Department of Biochemistry, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.

ABSTRACT

Prostate cancer (PCa) is the most prevalent cancer in men who live in developed nations. Even with the recent advancements in surgery, radiation, hormonal therapy, and medical management, medical treatment for advanced-stage PCa is being developed. Alternative strategies for reducing the prevalence of PCa include dietary cancer chemopreventive agents, it has been suggested that medicinal plants can treat cancer. Active composites of Capparis Cartilaginea (C. cartilaginea) are alkaloids, tannins, steroids, terpenoids, and flavonoids. This study aims to investigate the cytotoxic effect of C. cartilaginea fruit extract on the 22RV1 human prostate cancer cell line. Under an inverted light microscope, a dose- and time-dependent morphological alteration and cell migratory capacity were observed after exposure to the ethanol extract of C. cartilaginea fruit. The cytotoxicity of the C. cartilaginea fruit ethanol extract on the prostate cancer 22RV1 cells was performed by using the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. The effect of C. cartilaginea fruit extract on cell migration was assessed using an in vitro wound-healing scratch assay. The MTT assay showed that C. cartilaginea fruit extract had a significant cytotoxic effect on 22RV1 cells after 72 h treatment with a value of (0.088 ± 0.031) μg/mL. Following exposure to C. cartilaginea fruit extract, morphological changes in the cells showed that the extract had a remarkable impact on cell death, and the mortality correspondingly increased with the increase in concentration. The findings suggested that C. cartilaginea fruit ethanol extract might be used as a medicinal plant for treating prostate cancer.

Keywords: Prostate cancer, C. Cartilaginea, Wound healing, Flavonoids, Scratch assay, Fruit extract


Introduction

Prostate cancer (PCa) accounts for 6.6% of total deaths in males, making it the fifth leading cause of cancer mortality in men [1]. The Saudi cancer registry recorded 1739 cases in the Kingdom of Saudi Arabia during 7 years. The highest rate (10.1 per 100,000 men) was recorded in the eastern region of Saudi Arabia. The rate of PCa cases was 7.1 in Riyadh, 5.2 in Makkah, 1.4 in Jizan, and 2 in Najran [2]. In the Middle East, the incidence rate of PCa is significantly lower than in Europe and the United States [3]. In Saudi Arabia, PCa is the sixth most common cancer among men of all ages [4]. In 2018, the age-standardized incidence rate for PCa was 7.7 per 100,000 men, and the age-standardized mortality rate was 5.1 per 100,000 men, as estimated by the International Agency for Research on Cancer. When comparing Persian Gulf countries with European countries, the age-standardized incidence rate of PCa in Saudi Arabia is generally very low [2].

The early diagnosis of PCa by screening tumor markers is vital since the effectiveness of the ordinary treatment for PCa is useful for early-stage cancer when a tumor is localized within the prostate [5]. The common treatment of localized PCa with radical prostatectomy or radiotherapy may result in a serious negative impact on quality of life, including sexual dysfunction, urinary leakage, and compromised bowel function [6]. The choice of therapy is made based on clinical staging and the general health of the patient. Active investigation is typically used to manage localized PC a with low risk. More invasive therapy is necessary for intermediate and especially high-risk, diseases, for which radiotherapy or surgery is currently the standard treatment option, regardless of multimodal approaches. Androgen deprivation therapy (ADT), which focuses on androgen receptor signaling, is the go-to treatment for metastatic cancer [7]. PCa tumors grow very slowly before symptoms appear. As a result, there is a relatively long window for therapeutic interventions. Even a small delay in the disease’s progression could greatly enhance the quality of life for the patient. Dietary substance consumption can pose a significant clinical challenge in the quest to improve nontoxic chemopreventive strategies [8]. Plant-based drugs have lately received extensive research attention [9], largely due to significant advancements in organic chemistry, ground-breaking modifications to the extraction and purification processes, and a better understanding of the natural compounds found in plants. In addition, using plant sources as less expensive raw materials might help the pharmaceutical sector to create better drugs at significantly lower costs [10].

Capparis cartilaginea (C. Cartilaginea) (flower buds, fruits, and shoots) have been used in traditional medicine as effective treatments for numerous human diseases over the past thousand years. It can also be used as a drug, cosmetics, food, and animal nutrition [11, 12]. C. cartilaginea is widely distributed in the Arabian Peninsula (Saudi Arabia, and Yemen); North Africa (Egypt), Western Asia (Iraq and southern Iran); tropical Northeast Africa (Somalia and Sudan); tropical East Africa; and the Indian subcontinent [13]. Plants of this family are annual, perennial, and sometimes climbers or trees [14]. Six flavonoids isolated from C. cartilaginea are identified as kaempferol-3-O-rutinoside, quercetin-3-O-rutinoside, kaempferol 3- neohesperidoside, kaempferol 3-(2 G-rhamnosylrutinoside), quercetin 3-(2 G-rhamnosylrutinoside), and isorhamnetin-3-O-rutinoside [15]. Additionally, this genus includes non-nutritive plant chemicals known as phytochemicals, such as glycosides, tannins, flavonoids, alkaloids, saponins, unsaturated sterols, and coumarins [16]. Flavonoid and alkaloid compounds are used as drugs to prevent many diseases. Scientists have suggested that various types of cancer can be prevented and efficiently inhibited by these compounds [17]. According to a study carried out in an animal model, flavonoids and alkaloids can block the progression of the cell cycle, inhibit mutations, and prevent multistage carcinogenesis, thus breaking lipid peroxide chain reactions. They can also scavenge proxy radicals, superoxides, and hydroxides, as well as protect cells from X-ray damage [18]. Flavonoids have anticancer, antioxidant, anti-inflammatory, and antiviral properties, among other therapeutic advantages. Additionally, they have cardio-protective and neuroprotective effects [19]. Quercetin (3, 3′, 4′, 5, 7-Penta-hydroxy-flavone) Several cancers, including breast, lung, prostate, cervical, liver, and colon cancer, are prevented from spreading by quercetin, and it works by a variety of mechanisms, including cellular signaling, binding to cellular receptors and proteins, and inhibiting enzymes that activate carcinogens [20]. Glucosinolates (S-glucopyranosyl thiohydroximates) are a large group of secondary metabolites consisting of S­linked glucosides. The potential role of isothiocyanates as anti-carcinogenic agents has been thoroughly investigated as cytotoxic against a variety of cancer cell lines and is currently being utilized for the creation of new anticancer drugs [21].

Materials and Methods

Fruit Preparation

The fresh fruit of C. cartilaginea was collected from Wadi Molham, Wadi Abu Al-Haza, and Wadi Al-Quraine in Tuwaiq Mountains, Saudi Arabia in October 2018. The fresh fruit of C. cartilaginea was freeze-dried by freeze dryer (ilShin BioBase, Korea) at -60 °C under 5 mTorr pressure for 24 hr, then grind into powder by using Waring blender (USA). The powdered sample was kept at -80°C in an airtight container until required. C. cartilaginea fruit extract was ethanol extracted by using Al-Goufi and Sonbol methods [22]. Varying concentrations of the extract were prepared from stock solutions (0.5-20 μg/ml) and made up with complete RPMI‐1640 medium to a final volume of 100 μl. The criteria of cytotoxicity for the crude extract, which was established by the US National Cancer Institute, is IC50 <20 μg/ ml in the preliminary assay [23].

Cell Culture and Subculture Procedures

The prostate cancer 22RV1 cell line was maintained in RPMI-1640 medium supplemented with 10% (w/v) FBS and 1% (w/v) penicillin. Cells were grown in 25 cm2 tissue culture flasks in a humidified atmosphere containing 5% (v/v) CO2 at 37 °C. Once the cells reached 80% confluency, the medium was removed, and the cell monolayer was washed with PBS. Then, trypsin–EDTA (1 mL) was added to the flasks for 5–7 min to detach the cells' monolayer. The cells were occasionally observed under the inverted light microscope (Nikon Instruments, Melville, NY, USA) until the cell layer was dispersed. Then, a pre-warmed complete growth medium (3 mL) was added to the flasks followed by repeated gentle pipetting to split the cell clumps and inactivate the trypsin. After that, the cells were transferred to a 15 mL tube and centrifuged at 1500 rpm for 5 to 10 min. At this stage, the cell pellet was resuspended in a pre-warmed complete growth medium. Approximately 0.5–1 × 106 cells were sub-cultured into new 25 cm2 flasks that contained 8 mL of fresh medium. Morphological changes characteristic of cell death were observed using an inverted light microscope (Nikon Instruments, Melville, NY, USA).

Determination of Cell Viability and Proliferative Activity

The effect of the C. cartilaginea fruit extract on the growth of the prostate cancer 22RV1 cell line was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) calorimetric assay that measured the activity of mitochondrial dehydrogenase in living cells, which can convert pale yellow soluble MTT into an insoluble purple formazan product. Cells were grown at different densities (5000 cells/100 μL and 10,000 cells/100 µL) in 96-well microtiter plates (PermanoxTM, city) at 37 °C with 5% (v/v) CO2 overnight. Cells were treated with varying concentrations of C. cartilaginea fruit extract (0.5, 5, 10, 15, and 20 μg/mL). The culture plates were incubated at 37 °C with 5% (v/v) CO2 for 24, 48, and 72 h. Controls containing cells and media were included as positive controls.

After 24, 48, and 72 h of incubation, 10 μL of MTT (3 mg/mL) solution was added to each well and incubated at 37 °C with 5% (v/v) CO2. After 4 h of incubation, the supernatants were discarded. Subsequently, dimethyl sulfoxide (DMSO) (100 μL) was added to each well and vigorously mixed to dissolve the formazan crystals. Absorbance at 570 nm was measured with an enzyme-linked immunosorbent assay ELIZA reader (BioTek Instruments, Winooski, VT, USA). The percentage of cell viability was calculated manually using the Kamiloglu et al. formula [24]. The percentage of cell cytotoxicity was also calculated manually using the formula presented by Choudhury et al. [25].

Cells in a seeding density of 10,000 cells/well (and 5000 cells/well) were individually treated with different concentrations of C. cartilaginea fruit extract (0.5, 5, 10, 15, and 20 μg/mL) for 24, 48, and 72 h. Morphological changes in cells were observed 24, 48, and 72 h post-treatment.

An optimization of cell densities was performed, and the best representative concentration was found at densities of 10,000 cells/well. At a cell density of 10,000 cells/well, the number of viable cells in culture after treatment with varying concentrations of C. cartilaginea fruit extract over time was monitored.

Wound-Healing Scratch Assay

The effect of the C. cartilaginea fruit extract on the migration of the prostate cancer 22RV1 cell line was determined by using a wound-healing scratch assay [26]. Prostate cancer 22RV1 cells were grown to 75% confluency on 12-well plates and allowed to adhere at 37 °C with 5% (v/v) CO2 overnight. Consistently, shaped wounds were made using a sterile 10 μL pipette tip across each well, creating a cell-free area, based on the technique described by Cormier et al. [27]. Cultures were gently washed with PBS to remove loose cells. The cells were then exposed to varying concentrations of C. cartilaginea fruit extract (0.5, 5, 10, 15, and 20 μg/mL). Cells for the control conditions were also scratched, washed, and maintained in a culture medium supplemented with 10% FBS at 37 °C with 5% (v/v) CO2 positive control.

Immediately after the scratch, at 24, 48, and 72 hours, at least three images of the scraped area were captured using the DS-U3 digital camera controller (Nikon Instruments, USA) and imaging software (NIS-Elements version 4.0). Two independent experiments were performed, using two wells for each concentration. The same scratched area was selected for the measurements at each time of the study.

Measurement of Wound Area

The wound area was calculated by manually tracing the cell-free area in captured images of different periods using the ImageJ software (Fiji-ImageJ version 1.51 h) [28]. Firstly, the imaging software was calibrated to measure the area in microns instead of a pixel using a scale slide (Graticules LTD, Tonbridge, UK). Under normal conditions, the wound area will decrease over time, and the migration rate can be expressed as the change in the wound area over time. We measured the percentage of area reduction or wound closure. The closure percentage will increase as cells migrate over time [29]:

Wound closure % =At=0h-At=ΔhAt=0h × 100%

(1)

At=0h is the area of the wound measured immediately after scratching (t = 0 h).

At=Δh is the area of the wound measured h hours after the scratch is performed.

Statistical Analysis

The data were analyzed using GraphPad Prism version 8.2.1. (Prism 8). Significant differences between the treated groups and the control were determined by one-way ANOVA using Dunnett’s multiple comparisons tests. Results are expressed as mean ± standard deviation (SD) at a significance level of p < 0.05.

Results and Discussion

Effect of the C. cartilaginea Fruit Extract on the Cellular Proliferation of the 22RV1 Cell Line

The cytotoxicity effect of the C. cartilaginea fruit extract on the cellular proliferation of 10,000 cells/well of prostate cancer 22RV1 cell line, which was evaluated by the MTT assay, is presented in Table 1. At 24 h post-treatment, despite the increased resistance of the cells to C. cartilaginea fruit extract, there was a slight reduction in the number of viable cells in multi-well plates in parallel with the increase in C. cartilaginea fruit extract dose compared to untreated cells. As the concentrations of C. cartilaginea fruit extract increase from 0.5 to 20 µg/mL, the mean ± SD decreases. The percentage of viable cells decreases from 100% for the untreated cells to 63.86% for the treated cells with 20 µg/mL C. cartilaginea fruit extract. There was a dose-dependent decrease in the percentage of viable cells compared to the control (inverse relationship between cell cytotoxicity and concentration). Cell cytotoxicity increases with the increased concentration of the extract, starting from 4.66% to 36.14% for the concentration of 20 µg/mL C. cartilaginea fruit extract (Table 1, Figure 1). There was a significant correlation between the concentrations and cytotoxicity. As the incubation period increased to 48 h and 72 h, the number of viable cells in the control increased and the reduction in the number of viable cells in the treated wells became evident. Starting from the controls, the mean ± SD decreases with increasing the concentration of C. cartilaginea fruit extract. Furthermore, the percentage of viable cells decreases from 100% for the untreated cells to 17.54% for treated cells, and the cell cytotoxicity rises to 82.46 (Table 1, Figure 1). There were no significant differences in the mean ± SD of 0.5 µg/mL of C. cartilaginea fruit extract compared to controls. There was a significant correlation between the concentration of the C. cartilaginea fruit extract and cell viability. The cytotoxicity evaluation of the C. cartilaginea fruit extract against a prostate cancer 22RV1 cell line for incubation periods of 24, 48, and 72 h is shown in Figure 1.

 

Figure 1. Effect of the C. cartilaginea Fruit Extract on the Cell Viability 24hr, 48hr, and 72hr post-treatment, at a cell density of 10,000 cells/ well. Bars represent the mean ± SD. * is a significant difference at the level of p < 0.05.

 

Table 1. Effect of the C. cartilaginea fruit extract on the cell viability 24, 48, 72hr post-treatment, at a cell density of 10,000 cells/ well.

24-hour post-treatment

Concentrations

Mean ± SD

C%

Cell Cytotoxicity %

Adj. P-Val

Control

0.4637±0.5087

100

-

-

0.5 μg/ml

0.4421±0.03840

95.34

4.66

0.6667

5 μg/ml

0.3818±0.03367

82.34

17.66

* 0.0003

10 μg/ml

0.3522±0.02575

75.95

24.05

* <0.0001

15 μg/ml

0.3212±0.03203

69.27

30.73

* <0.0001

20 μg/ml

0.2961±0.04991

63.86

36.14

* <0.0001

48-hour post-treatment

Concentrations

Mean ± SD

C%

Cell Cytotoxicity %

Adj. P-Val

Control

0.6857±0.09010

100

-

-

0.5 μg/ml

0.6192±0.1304

90.30

9.70

0.2969

5 μg/ml

0.4863±0.07583

70.92

29.08

* <0.0001

10 μg/ml

0.4293±0.06511

62.61

37.39

* <0.0001

15 μg/ml

0.3679±0.05027

53.65

46.35

* <0.0001

20 μg/ml

0.2272±0.04441

33.13

66.87

* <0.0001

72-hour post-treatment

Concentrations

Mean ± SD

C%

Cell Cytotoxicity %

Adj. P-Val

Control

0.9101±0.1257

100

-

-

0.5 μg/ml

0.7492±0.2089

82.32

17.68

0.1568

5 μg/ml

0.5214±0.3170

57.29

42.71

* <0.0001

10 μg/ml

0.3010±0.02190

33.07

66.93

* <0.0001

15 μg/ml

0.2578±0.01255

28.33

71.67

* <0.0001

20 μg/ml

0.1596±0.03138

17.54

82.46

* <0.0001

Data are expressed as mean±SD, C%: cell viability, and Adj. P-Val: Adjusted. P-value, SD: standard deviation, *P<0.05

Evaluation of Morphological Changes Upon Treatment with C. cartilaginea Fruit Extracts

The morphological alteration of prostate cancer 22RV1 cell lines after exposure to different concentrations of C. cartilaginea fruit extract (0.5, 5, 10, 15, and 20 µg/mL) over time was observed under an inverted light microscope at 20× magnification. The microscopic observations revealed that the C. cartilaginea fruit extract had a significant effect on cell death, and the mortality rate correspondingly increased with the increase in concentration. After 24 h of incubation, cells exhibited typical proliferation in the absence of C. cartilaginea fruit extracts. Cells presented intact morphology, uniform size, clear outline, and vigorous growth, all of which grew adherently. No contact inhibition was observed between cells, indicating that cells were in a very healthy state (Figure 2A; 24 h). Treated cells with 0.5 µg/mL C. cartilaginea fruit extract displayed characteristic features similar to untreated cells. There were no noticeable changes in cell morphology (Figure 2B; 24 h). Cells treated with 10, 15, and 20 μg/mL C. cartilaginea fruit extracts were heterogeneous in appearance, with some cells demonstrating a tight adherence to the tissue culture plate and others remaining less adherent and round. The reduction in the size of the cells could be a feature of early necrosis (Figure 2D-2F; 24 h).

After 48 h of incubation, cells under control conditions appeared healthy and produced up to ~70% confluency (Figure 2A; 48 h). The cells look normal and have a bright color, and clear boundaries, as well as branching and spreading on the surface of the tissue culture plate (Figure 2A; 48 h). For treated cells with 0.5 and 5 µg/mL C. cartilaginea fruit extracts, there was no major presence of cell shrinkage, rounding, and partial detachment (Figure 2B and 2C; 48 h). Few cells exhibited morphological changes and their shape changed to circular (Figure 2B and 2C; 48 h). On the other hand, the cells indicated the most prominent effects as they began to leach from the tissue culture plate and became flat and shrunken after incubation with 10 μg/mL C. cartilaginea fruit extracts (Figure 2D; 48 h). After exposure to 15 μg/mL C. cartilaginea fruit extracts for 48 h, necrosis becomes evident. The majority of cells experienced a loss of their original morphology and had a swollen appearance (Figure 2E; 48 h). Treating cells with 20 μg/mL C. cartilaginea fruit extracts displayed clearer changes in cell morphology comparable to those observed in cells treated with 15 μg/mL C. cartilaginea fruit extracts (Figure 2F; 48 h).

At 72 h of incubation with C. cartilaginea fruit extracts, cells under control conditions appeared healthy and produced up to ~90% confluency. The cells appear to be over con-fluent, branching and spreading on the surface of the tissue culture plate since cells compete for the rest of the nutrients to survive (Figure 2A; 72 h). At a concentration of 0.5 μg/mL C. cartilaginea fruit extracts, some cells had characteristic features of cell shrinkage and partial detachment (Figure 2B; 72 h). The cells demonstrated morphological changes compared to the control. Cell shrinkage and losses were adherent to the plate after incubation with 5 μg/mL C. cartilaginea fruit extracts (Figure 2C; 72 h). At a concentration of 10 μg/mL C. cartilaginea fruit extracts, dead cells leached from the culture plate and filled the media, revealing an interruption in the extracellular matrix, as well as inhibition of cell-to-cell contact (Figure 2D; 72 h). Although wells were treated with 15 and 20 μg/mL C. cartilaginea fruit extracts, marked by the complete circularization of almost all cells and cloudy appearance (Figure 2E and 2F; 72 h). The cells exhibited significant morphological changes, such as a decrease in cellular volume, rounding, and shrinkage. Cells dramatically decreased in number, with almost no presence of viable cells (Figure 2E and 2F; 72 h).