Abstract
The current study examined the potential therapeutic advantages of catechin, either alone or in combination with docetaxel (DTX), against PC-3 prostate cancer cells. Following the MTT assay’s determination of the IC50 concentrations, the cell lines were subjected to 48 h of treatment in the following protocol: untreated PC-3 control cells, docetaxel treatment, catechin (Cat) treatment, and DTX + Cat therapy at a ratio of 1:1. Treatments with DTX and Cat significantly decreased the number of viable cells in PC-3 cells in a dose-dependent manner. Additionally, the combo treatments caused the highest cytotoxicity compared with the other treatments. Also, when DTX and Cat were combined, they caused a significant synergistic effect (CI < 1) (combination index < 1). Furthermore, it was demonstrated that all treatments increased the expression of BAX, Caspase-3, and P21 mRNA in PC-3 cells while decreasing that of BCL-2 mRNA. The highest proportion of overexpression was observed in the combo therapy. A greater proportion of early and late apoptotic cells were caused by the combined treatment than by > DTX > Cat, according to flow cytometry. DNA damage in PC-3 cells was detected using the comet assay, and values of DNA tail, tail length, and tail moment increased considerably with increasing treatment dose. According to this study, Cat is effective against PC-3 cells when used in conjunction with DTX; by causing apoptosis, it enhances DTX’s chemotherapeutic capability in cancerous cells.
Introduction
Prostate cancer (PC) is one of the malignant diseases that is becoming more common among men.1 According to epidemiological statistics, prostate cancer is the 4th greatest malignancy after colorectum cancer in both genders as well as the eighth most prevalent reason of dying in both sexes, with an estimated number of 1,467,854 new cases and 397,430 deaths globally. It represented the second malignancy after lung cancer in men according to Globocan 2022. Its incidence is increasing among male patients due to smoking.2
Chemotherapy, surgery, and natural products are approaches for cancer treatment.3 Despite it many drawbacks, chemotherapy is the most widely used cancer treatment. Natural chemicals can be utilized as a stand-alone treatment or in combination with conventional chemotherapeutic medications.4 Many plants and animal extracts have exhibited diverse biological activities such as immunological potentiating and anticancer activities with minimum or no harmful side effects, demonstrating a high level of interest in moving from chemo to biotherapies as a cancer treatment.5 Alkaloids, terpenes, phenols, and flavonoids are just a few of the natural compounds that have been demonstrated to destroy cancer cells in clinical trials.6
V. vinifera L. (black grapes) is a common crop in the Mediterranean region, and it is a rich source of bioactive chemicals that help the body’s defense processes.7 This blooming plant is indigenous to Central Europe, the Mediterranean basin including Egypt; Southwest Asia stretches from Morocco and Portugal to southern Germany in the north and northern Iran in the east. Cat is the most abundant polyphenolic compound in black grapes, and has shown therapeutic effects against multiple types of cancers in vitro and in vivo, it is a potential cancer treatment.8
Green tea, red tea, and red grapes contain catechin, a bioactive polyphenol compound that has strong antioxidant capabilities and is used to lower the risk of several diseases due to its antiviral, antioxidant, and antitumor effects.9,10 Epigallocatechin gallate (EGCG) is the most prevalent catechin in red tea, green tea, and grapes although it contains many other beneficial compounds as well. In addition to being efficient scavengers of reactive oxygen species in vitro, tea catechins and polyphenols may also have indirect antioxidant effects by influencing transcription factors and enzyme activity.11 Catechins undergo extensive and fast metabolism, which highlights the significance of proving their antioxidant action in vivo. Catechin derived from green tea demonstrated significant effects against various cancers in cellular and animal cancer models, including lung, ovarian, liver, bladder, skin, and prostate cancer.12 Using natural compounds in combination therapy is a promising approach.
Apoptosis is triggered by the primary pro-apoptotic gene p53 (tumor suppressor), which also causes DNA repair and initiation of other cell cycle checkpoints, like the G2/M and G1/S phases. Bcl-2 gene families are influenced to trigger apoptosis, while Bax, a part of the Bcl-2 protein class, is one of the downstream mediators of p53-dependent apoptosis, through the elimination of cytochrome C from the mitochondria’s inner membrane gap. It initiates the caspase pathway upon translocation from the cytosolic to the mitochondria, which is the penultimate stage of apoptosis commitment.13
Docetaxel (DTX) is an anti-tumor produced from the needles of Yew trees; it belongs to the toxoids class of anti-cancer medications.14 Its comprehensive antitumoral action in the treatment of a variety of tumors, such as stomach, prostate, connective tissue, and breast cancer, is responsible for its effectiveness as a therapeutic drug.15 This prevents the normal assembly of microtubules to the mitotic spindle, which stops the cell’s cycle during the G2/M phase. It also inhibits the gene expression of Bcl-2, a reduced apoptotic gene that malignant cells frequently overexpress, enabling them to live, if this gene is downregulated, cancer cells can undergo apoptosis easier.16
Recently, combination therapy refers to the treatment of a disease with two or more medications.17 Combination index (CI) analysis represents drug combinations can produce a variety of consequences, such as functional antagonism, enhanced drug toxicity (CI > 1), and synergistic (CI < 1) or additive effects (CI =1).18
Accordingly, the current work aimed to discover the efficacy of Cat and DTX combination therapy compared to the efficacy of each medication separately. Furthermore, using biochemical and molecular genetic parameters, the work assessed the fundamental processes that give rise to anticancer effects of Cat, DTX, and the combinations, as well as whether using genetic data could help better customize treatments for prostate cancer.
Material and method
Chemicals
Cat standards including Cat, EC, GC, EGC, GCG, and EGCG, also internal standard l-tryptophan, were obtained from Sigma-Aldrich Co. [St Louis, MO, US]. HPLC grade solvents comprising ethanol, methanol, and acetonitrile were obtained from Merck Millipore [Darmstadt, Germany]. Formic acid was obtained from Sigma Aldrich Co. A Milli-Q water purification system from Merck Millipore was used to create deionized water. TAXOTERE (Sanofi-Aventis U.S. LLC Bridgewater, NJ 08807A) was obtained as a sterilized, light yellow to browned yellow solution (20 mg of DTX in each ml.
Preparation of grape seed extract and Catechin verification
The common grape, V. vinifera L was obtained fresh from Janaklees farm, East Alexandria- Northern Egypt. For extraction, the grapes were mashed into tiny pieces and then ground within a volume equal to double the weight of the tissue in 96% ethanol. Homogenized tissues were rinsed in ethanol for 48 h at RT and then filtered twice. The ethanol was removed using a rotary evaporator, and in a vacuum oven, the residue was dried for 48 h at 40 °C. The extracted powder was then stored at 20 °C till needed. The extract’s purity was determined using mass analysis chromatography according to the mass spectroscopic analysis performed employing the NY | USA Advion compact mass spectrometer (CMS). Before the injection, the powder was identified on a TLC plate after being dissolved in methanol. Typically, the fragment was carried out using type ESI and APCI at a mass ranging from 100 to 1,200. Cat extracted using liquid chromatography with high performance (HPLC-DAD) Waters 996 photodiode array detector installed in a Waters 2,690 Alliance HPLC device. Three HPLC columns, including Gemini C18 110A column length (CL) = 250 mm, internal diameter (ID) = 4.6 mm, particle size (PS) = 5 μm, Vydac 201TP54 C18 (CL) = 250 mm, (ID) = 4.6 mm, (PS) = 5 μm, and TSK gel ODS-80Tm C18 (CL) = 250 mm, (ID) = 4.6 mm, (PS) = 5 μm, were acquired from Phenomenex Co. [Torrance, CA, US], Vydac Co.[Hesperia, CA, USA], and Tosoh Co. [Tokyo, Japan], respectively. An internal standard of 1,000 μg/mL of l-tryptophan was dissolved in a 50% ethanol solution for quantitative analysis. The different standards’ curves were then determined by graphing the concentration ratio (standard versus internal standard) against the area ratio (standard versus internal standard). A Microsoft Excel software system was used.
Cell culture and determination of IC50 by MTT assay
The human PC3 prostate cancer cell line was acquired from Vaccera, Co., Giza, Egypt, which was initially acquired from the American Type Culture Collection [ATCC, CRL-1435]. Acid phosphate levels are low and testosterone-5-alpha reductase activity was confirmed poor in these cells. Monolayer cultures of the cells were maintained at 37 °C in a 5%(v/v) CO2 atmosphere using Roswell Park Memorial Institute medium, RPMI-1640 (PAA – cell Culture Company, Germany through Immune, Poland). The media was enhanced with 10% FBS, 4.0 mmL glutamine, 100.0 U/mL penicillin, and 100.0 mg/mL streptomycin. The cell line was kept in media includes DMEM with 10.0% of (v/v) FBS and 1.0% (v/v) penicillin/streptomycin then incubated at 37 °C with 5.0% CO2. The cells were cultured at a concentration of 1.0 × 104 cells/ml into 96 well plates then it was incubated for a 24 h under standard conditions. Adding 5.0 mg/mL of the MTT solution to each plate, then measured at a wavelength of 630 nm using an ELISA microplate [Bio-Rad microplate reader, Japan]. The cell viability was determined using the following equation: Percentage of viable cells = [Abs s/Abs c] 100. The OD (or optical density) of every well was determined using spectrophotometry at the wavelengths 545.0 and 540.0 nm. The values of the IC50 were determined using sigmoidal concentration-response curve fitting models (sigma plot program).
The experimental plan
Cat and DTX were added to the media at concentrations 1,000, 800, 600, 400, 200, and 0.0 μg/mL for Cat, and 1.0, 0.8, 0.6, 0.4, 0.2, and 0.0 μg/mL for DTX to determine the IC50 for every drug. Three groups of PC-3 cells were treated: G1: Non-treated control cells (vehicle alone) (used as positive controls), G2: Docetaxel treatment at IC50 and G3: Catechin treatment at IC50 and G4: v/v combinations of DTX (IC50) and Cat (IC50).
Drug combination index study
The two medications were added to the media at different (v/v) concentrations of 1:1, 1:2, 2:1, 1:4, 4:1, 1:9, and 9:1 (DTX: Cat).17 The combination index (CI) was calculated using the data obtained from the MTT experiment to examine the synergistic antiproliferative effects of DTX and Cat. The concentration-response curves used in this combination study plot the percentage of unaffected cells vs drug concentration using the Chou & Talalay approach.18 A synergistic impact is indicated by CI values less than 1, an antagonistic effect is shown by values greater than 1, and an additive effect is indicated by values equal to 1.
Apoptosis detection methods by flow cytometry
Assay for the Annexin V-FITC Staining: Mammalian DNA has been stained for flow cytometry following.19 After treating PC3 cells with an IC50 dose, apoptosis was assessed using the Fluorescence activation of the cell sorting (FACS) caliber flow cytometer (Becton Dickinson, Sunnyvale, CA, USA) equipped with emission filters of 488 nm. This was achieved by determining the cell surface of phosphatidylserine through apoptotic cells with the Annexin V-FITC/PI apoptosis detection kit (the BD Pharmingen).
Analysis of the cell cycle via flow cytometry
The Annexin V-FITC/PI apoptotic identification kit (the BD Pharmingen) was used to determine the amount of phosphatidylserine on the cell surface of apoptotic PC3 cells. The results were then analyzed via a fluorescence Activation The cell Sorting (FACS) caliber flow cytometer (Becton Dickinson, Sunnyvale, California, the United States) was equipped with 488 nm emission filters. Briefly, PC3 cells were treated with IC50 dosages and then placed into propidium iodide solution for 15.0 min before being analyzed on a flow cytometer that was calibrated for FACS. After 1 h of staining, the samples were processed through the flow cytometer, and the stained samples were kept at room temperature throughout the night. The stoichiometric dyes which attach proportional to the quantity of DNA that exists in the cell are measured by the flow cytometer.20
Comet assay
A 10.0 μL aliquot of cell suspension, including about 10,000 cells, was mixed with 75.0 μL of 0.50% of LMA and distributed in cold lysis buffer (freshly added 10.0% DMSO and 1.0% of Triton X- 100, respectively, were added after pH adjustment). The slides were incubated in a lysis buffer for 24 h at 4 °C in a light-protected condition and then placed in an electrophoresis buffer for 20 min to unwind DNA. In the dark electrophoresis was performed for 20 min at 300 mA with 25.0 volts (0.90 Volts/cm). The slides were washed in a neutralization buffer after electrophoresis, fixed in 100% ethanol for 5 min to air dry then kept at 4 °C until scoring. Ethidium bromide fluorescent dye was applied to the slides before scoring.
Slide scoring
The degree of DNA movement in each sample was evaluated by photographing 50 cells at 400 × magnification with a fluorescence microscope camera (Leica, Germany). The properties of the comet were studied using Kinetic Imaging Ltd’s Komet 5 image analysis program (Liverpool, UK). The amount of damaged DNA in cells was assessed by three comet parameters: Tail length (μm) is the distance that runs from the end of DNA head to the endpoint of DNA migrating. DNA content (%) in the tail is calculated when; the degree of intensity of each tail pixel is divided by the total intensity of all comet pixels. Tail moment is calculated as tail length multiplied by the percentage of the total DNA in the tail [Tail moment = (Tail length × % DNA in tail) /100].
Semi-quantitative RT-PCR analysis
1.0 μg of extracted RNA, 1 μL of the random hexamer primers, and 12.0 μL of the DEPC-treated water were mixed, rapidly vortexed, and then incubated at 65 °C for 5 min. After using the RevertAid H Minus Reverse Transcriptase (200 U/μL) to create the copy DNA, the samples were gently combined, centrifuged, and incubated at 25 °C for 5 min then at 42 °C for 60 min. Reaction products were frozen at −80 °C. GAPDH-specific control primers were used to confirm the synthesis of cDNA. Following PCR, 10 μL of the RT-PCR product was transferred onto a 1.50% agarose gel, and a system for gel documentation was used to visualize the bands. Using newly produced cDNA as PCR templates, Bcl-2, Bax, Caspase-3, and CDKN4 gene expression was measured (the primers’ sequences are shown in the Supplementary Table 1). 25.0 μL of Qiagen Taq green master mix, 4.0 μL of cDNA, 2.0 μL of forward primer, 2.0 μL of reverse primer, and 17.0 μL of nuclease-free water had been pre-denaturized at 94 °C for 3 min. Non-reverse transcription RNA molecules have been used to ensure that no genomic DNA was present. To check for reagent contamination, a negative control was added without the template.
Schematic diagram of materials and methods.
The PCR was duplicated, and the band intensities were densitometrically analyzed using a gel documentation system. To compare the levels of mRNA expression of Bcl-2, Bax, caspase-3, as well as CDKN4 (p21) to GAPDH, the average ratio (mean) of band intensity standard deviation was calculated after that plotting versus test concentrations and time intervals. For statistical significance analysis, the one-way t-test has been used. GAPDH was used as an internal control. As previously mentioned, the thermal cycle parameters, melting curve temperatures, and relative expression calculation utilizing 2−ΔΔCt were executed.21
RNA isolation
Following the manufacturer’s procedure, RNA was extracted from treated and untreated cells of PC3 after 24 h of treatment with a Qiagen kit (One Step RT-PCR kit, Qiagen, Valencia, CA). The extracted RNA was kept at −80 °C; its concentration and purification were assessed by diluting it with purified water and then measuring the optical density spectrophotometrically at 260 and 280 nm. Here is how the concentration and purification of RNA have been calculated: Conc of the extracted RNA (μg/mL) = [A260 X dilution factor X 40 RNA purity] = [A260 /A280].
Results
Qualification and development of the HPLC-DAD technique
The HPLC-DAD technique was created and permitted to evaluate the phenolic acids and flavonoid content in comparison to standards detected at 280 nm in black grapes extract. At the same time, the entire analytical variable, involving the calibration curves and the linear range, was optimized. Analyses were performed in triplicate and catechin, rutin, quercetin, chlorogenic acid, gallic acid, caffeic acid, ellagic acid, and kaempferol have been taken as standards. The chromatographic separation of the phenolic acids and flavonoids in black grapes extraction detected the presence of Cat, caffeic acid, quercetin, and Kaempferol. Readings at 520 nm were also taken and demonstrated the absence of anthocyanins. The chromatographic identification results, which involve the analytes’ retention times (RTs), and their respective maximum absorption wavelengths (λ, nm) are presented in Figs. 2 and 3, respectively. The flavonoid content is summarized in Supplementary file 2.
HPLC chromatography of A) Combined standard solution and B) Grapes extraction.
Auto-scaled chromatography of A): Combined standard solution and B) Grapes extraction.
Thin layer chromatography—Mass spectrometry (TLC/Ms)
The data of soft ionization for the 2 molecules of catechin produced by TLC–Ms the chromatogram of catechin molecules is shown in Fig. 4(A) and Supplementary Table 3, which represents low degradation percent due to the stability of catechin. Figure 4(B) shows the molecular weights of catechin molecules.
A). TLC chromatogram of catechin. B) Mass spectrum of catechin molecules with molecular weights 290.4 and 580.4.
Cell viability data by MTT assay
Figure 5(A and B) displays the cells vitality of PC3 cell lines after treatment with various concentrations of DTX or Cat for 48 h respectively. In a dose-dependent manner, the percentages of living cells decrease as DTX and Cat concentrations increase. It has been calculated that the inhibitory concentration (IC50) of DTX to PC3 cells was 0.6 μg/mL, and that of Cat was calculated to be 400 μg/mL.
The percentage of viable cells at a 48-h incubation period using different concentrations of DTX (A) or cat (B) was measured.
The combination treatment of different IC50 concentrations of DTX and cat
The outline of the interaction between drugs was recognized utilizing the standard non-constant isobolograms or a CI plot after PC3 cells have been treated during 48 h using a combination of drugs with IC50 ratios (1:1, 1:2, 2:1,1:4, 4:1, 1:9, and 9:1) (DTX: Cat). Using the combination index formula (CI) of Chou and Talalay18, it is found that DTX combined Cat with 1:2 IC50 concentration ratios have an overall highest synergetic prostate cancer inhibitory effect with a combination index < 1 (CI = 0.84). The data also showed that the combinations of 1:1, 1:4, and 1:9 volumes are also synergistic (CI = 0.97), but with a lesser significant value (Fig. 6). The 2:1 combination was antagonistic (CI > 1; 2.41), while the 4:1; 9:1 combination were beyond detection level.
The combination index (CI) for analyzing DTX or cat was estimated by the Chou and Talalay (1984) technique. A) The effect of dosage curves showing combined dosage on the Y axes referring to the respective values of two medications; B) A combination index scheme indicating a strong synergistic interaction (CI < 1) to 3 combinations; C) The diagram of log dose-reduction index (DRI) for non-constant DTX + cat combined. D) A diagram for the median effect level for DTX and cat and (DTX + cat combined). Fa: A standard of default effect.
Bax, Bcl-2, Caspase-3, and CDKN1 mRNA gene expression in treated PC3 cells
Figure 7 demonstrates the normalized levels of mRNA expression of Bcl-2, Bax, Caspase-3, and CDKN1 in all groups measured with qRT-PCR relative to GAPDH internal control after 48 h. All treatments significantly reduced the Bcl-2 expression. Bax expression values were significantly overexpressed after treatment, particularly the combination in G4 over the other treatment groups. Similarly, Caspase-3 expression values display significant overexpression after treatment with the combination group above the other treatments. CDKN1 also showed a significant over-expression as compared with the other treatment groups.
The qRT-PCR analysis showing the normalized mRNA expression of Bcl-2 (A), Bax (B), Caspase-3 (C), and CDKN1 (D) in all experimental groups. *: Significant as compared vs control (G1) (P < 0.01). a) Significant vs. G3 and G4 (P < 0.5). b) Significant vs. G2 and G3 (P < 0.01). RQ: Fold change by real-time qRT-PCR relative to GAPDH.
Combination therapy promotes late apoptosis in PC3 cells
Using flow cytometry and dual staining on cells with Annexin-V/PI, it has been demonstrated that therapies with DTX, Cat, DTX + Cat reduced the percent of viable cells to 64.6%, 65% and 28.4%, respectively. The least reduction in viability of cells was indicated in the cells that received dual treatment with DTX + Cat (Group 4). Flow cytometric analysis data of the percent of apoptotic cells in treated and non-treated PC3 cells are demonstrated in Fig. 8. Non-treated PC3 cells normally exhibited a very low percentage of early and late apoptotic cells (0.1 and 0.1% respectively). When PC3 cells were treated with DTX, Cat, or v/v combination of both, the percentages of late apoptotic cells significantly (P < 0.001) raised to 28.3% when treated with DTX, 22.1% when treated with Cat, and 67.7% when treated with v/v combination of both drugs. The percentages of early apoptotic cells were non-significantly increased after treatment with DTX and Cat or the combination of both drugs, was 1.7%, 1.6%, and 2.6% respectively.
A) Flowcytometric analysis data showing average percentages of early and late apoptotic cells in DTXtreated cells, cat, and v/v combination of DTX + cat. All treatments mostly induced apoptosis particularly the combination treatment; *: Significant vs. un-treated control cells at P < 0.01; a: Significant vs. G2 and G3 at P < 0.001. B-E) Instance flow cytometry dot plot log graphs indicating the percent of early and late apoptotic cells in treated and untreated PC3 prostate carcinoma cells; B) PC3 un-treated cells, C) Cat treatment, D) DTX treatment, E: Combination treatment. Inside each dot plot log figure, the upper left square is % of necrotic dead cells; lower left: % of viable cells; upper right; % of late apoptotic cells; lower right: % of early apoptotic cells.
Cell cycle analysis
Analysis of the cell cycle (Fig. 9) demonstrated that cells treated with the combination of Cat + DTX displayed the lowest S-phase fraction and the greatest Sub-G1 cell population. Furthermore, DTX, Cat, and DTX + Cat treatment resulted in the G0/G1 phase arrest of 62.5%, 56.5%, and 74.8% of cells, respectively.
Flow cytometry is used to analyze the cell cycle and to determine the percent of apoptotic cells (in the sub-G1 phase). A–D) represent flow cytometry histograms of propidium iodide-stained cells displaying distinct cell cycle stages of untreated cells and treated cells with DTX, cat, and DTX + cat, respectively. E) Illustrates the mean values of the cells during sub G1, G0/G1, S, and G2/M phases.
Cytotoxicity by comet assay
Figure 10 shows the potential DNA damage measured by the DNA percentage in the comet tail, the %DNA in the tail, and the tail moment. All treatments triggered the destruction of DNA by a significant increase in tail length, especially the combination treatment (P < 0001). In addition, all treatments notably increased the percent of DNA in the tail and the tail moment; in contrast to the untreated control values, the combination treatment yielded the highest significant effect levels (P < 0.001).
A-C) Comet assay data in all studied groups. A: Tail length; B: Tail moment data; C) %DNA in tail. DG: Photomicrographs of comet assay showing DNA toxicity in untreated cells D): Un-treated control PC3 cells; E): DTX treated group. C): Cat-treated group. D) Combination treated group.
Discussion
The most prevalent bioactive compounds found in fruits, vegetables, seeds, and other foods are called polyphenolic substances. They have a variety of uses in the prevention and cure of diseases, notably cancer. The flavonols and flavones in green tea include myricetin, quercetin, apigenin, and kaempferol.22 Because of their significant anticancer properties, catechin and the isoflavone genistein have cytotoxic, proapoptotic, anti-inflammatory, antiangiogenic, antimetastatic, and anti-invasive characteristics. Several experimental studies have demonstrated the strong potential of these substances in fighting against cancer, including in vitro cell cultures and rodents.23 Numerous experimental works based on the evaluation through in vitro and in vivo assays have confirmed the therapeutic application of many natural compounds, which have been later included as part of approved treatments, including in anticancer agents.24
In this investigation, the variety levels of Cats in grapes were evaluated through mass spectrometry and high-performance liquid chromatography. According to the results, eight Cat standards were successfully separated in 25 min. The proliferation of PC3 tumor cells may be suppressed by Cat extract, with a half-maximum inhibitory concentration of 400 μg/mL.
Our study showed that Cat inhibits the growth of prostate cancer cell PC3. This result is in line with,12 who studied the anti-cancer functions and the molecular mechanisms of catechin green tea, especially EGCG, which is found in various animal cancer models and in vitro experiments using different types of cancer cells. DTX has an anticancer effect on PC325 reported that DTX is the first line of treatment for this tumor. In the current investigation, we have confirmed that the Cat treatment regimen (dosage and duration) with or without DTX exhibits strong anticancer effects.
In the current study, we found that Cat could inhibit prostate tumor cells, increasing apoptosis significantly in terms of the apoptosis induced by DTX. The MTT test was applied to measure the IC50 values of DTX solution, Cat solution, and their combination at various ratios in PC3 cells to examine any potential synergistic effects of DTX and Cat. A combination of DTX and Cat at a ratio of 1:2 may improve the effectiveness of each drug’s ability to inhibit cell proliferation which had stronger synergistic effect than others.
In this study, the analysis of DNA fragmentation in PC3 cells showed that the percentage of fragmented DNA increased with Cat treatments and its combination compared to untreated cells. The DNA extracted from all the treated cells displayed DNA damage when compared with the control. The combination of DTX and Cat was more potent and destructive to DNA than the Cat treatment and higher than in the DTX treatment. These results are consistent with other studies that show the flavonoid’s ability to cause DNA fragmentation.26
The induction of apoptosis is a fundamental mechanism that underlies the anticancer therapeutic activities of several medications.27 Traditional cancer therapies like chemotherapy and radiation largely target cancerous cells by inducing apoptosis programmable cell death.28 Furthermore, multiple studies showed that Cat caused apoptosis in several cell lines.29 In vitro studies on several cancer cells, including breast, melanoma, and cervix, revealed that tea catechins inhibited growth and triggered apoptosis.30
BAX can induce the formation of the mitochondrial pore, which releases cytochrome c, thus activating the caspase cascade, so this observation is in agreement with previous studies in which BAX induced mitochondrial membrane potential disruption.31
In the current work, the genetic expression of the antiapoptotic precursor, BCL-2, and the proapoptotic precursor, BAX gene, was determined by qRT-PCR analysis. When compared to GAPDH-normalized control PC3 cells, it was discovered that the BCL-2 gene’s average normalized expression (RQ) was downregulated by around 0.04 fold in group 2 treated with DTX, lower by 0.14 fold in group 3 treated with Cat, and lower by 0.12 fold in group 4 treated with DTX + Cat combination. There was a statistically significant difference (P < 0.05) in the RQ values of groups 2, 3, and 4.
The BCL-2 gene is a member of a class of protooncogenes that inhibit apoptosis to increase cell survival. One of the BCL-2 gene family’s apoptosis-promoting members is known as the BAX gene. The distinctive characteristic of the BCL-2 protein is its ability to increase the number of cells by inhibiting apoptosis. Its ability to prevent death also appears to be proportional to its expression level. In contrast, the BAX protein causes apoptosis when it is overexpressed in a range of eukaryotic cells. BCL-2 proteins are evolutionarily conserved regulators of apoptosis. The BCL-2 class is comprised of two proapoptotic and antiapoptotic family members.32
The results of this study demonstrated that the BAX gene was detected and markedly decreased compared with the control group. On the other hand, when compared to RQ levels of normalized control non-treated PC3 cells, relative expression of the BAX gene was found to be 2.01-fold higher in group 2 treated via DTX, 1.4-fold higher in group 3 treated via Cat, and 2.8-fold higher in group 4 treated via DTX + Cat. According to these findings, Cat has significantly raised the potency of DTX molecular processes, which downregulates the BCL-2 gene and overexpresses the BAX gene.
These expected data are in line with previous data on the behavior of BCL-2 and BAX genes during the apoptotic pathways. Several substances, including P53 and the BCL-2 protein class, regulate the process of apoptosis. Concerning events involving DNA damage, the natural form of the P53 protein performs a physiological role as a transcription component that binds DNA and may promote apoptosis.33
CDKN4 regulates the progression of the cell cycle during the G1 and S phases. Furthermore, it was demonstrated that CDKN4 is an inhibitor produced from senescent cells that can induce cell senescence. CDKN4 is mainly based on two elements: the stimulus supplied and the kind of cell. Arrest of the cell cycle happens to repair damaged cells. The activation of CDKN4 by P53 and the concurrent inhibition of CDKs are thought to be essential for CDKN4’s tumor-suppressive function, as CDKN4 has also been demonstrated to regulate P53-induced G1 cell cycle arrest.
In our study, the P21 (CDKN1) gene was found to be upregulated by about 1.4-fold in group 2 treated with DTX, 1.19-fold in group 3 treated with Cat, and 2.49-fold higher in group 4 treated with DTX + Cat combination. These results were in line with,34 who have reported that induction of p21 through RNAa inhibited prostate cancer cell growth both in vitro and in vivo.34 On the other hand, the DTX and v/v combination-treated groups showed a significant (P < 0.05) up-regulation of CDKN4 expression in comparison to the negative, non-treated control group. The treatment with the combination has significantly exerted a higher and these results lined with,35 who said that the CDKN4 gene is downstream of P53; as a result, when P53 is subjected to various challenges, including DNA damage and oxidative stress, its activity will be up-regulated, increasing the expression of CDKN4.
Caspase-3 is one of the main apoptosis executors among these apoptotic caspases.36 Death receptors and the mitochondrial pathway, the two major apoptotic pathways, are shared by Caspase-3 activation37 therefore, in our study, when comparing the DTX and v/v combination-treated groups to the negative, non-treated control group, it was discovered that the Caspase-3 gene was considerably up-regulated (P < 0.05). The apoptotic process, also known as “programmed cell death,” is primarily carried out by a group of cysteine proteases identified as caspases. Caspases cause DNA breakage, chromatin condensation, and other morphological changes that are typical of an apoptotic cell, including cell shrinkage.38
We noticed that more cells were in the G1 phase in all groups, but the Cat and Cat + DTX groups showed this to be more significant. In terms of phase G2, the Cat, and Cat + DTX groups showed lower results, and as was predicted, the lower percentages discovered in phase S showed little to no variation between groups. These findings are significant since tumor cells are well-recognized to be more susceptible to the negative effects of chemotherapy during the G0 and G1 phase cycles.39 The discovery that Cat-treated groups likewise displayed a lower percentage of these in phase S suggests that PC3 cells have a reduced capacity to divide.
The comet assay results in this study showed a clear dose–response interaction, with the percentages of tail DNA (TDNA), tail length (TL), and tail moment (TM) higher according to rising radiation dosage. By measuring 50 comets in micrometers, the comet tail length (TL) was determined. By multiplying the comet tail length by the comet tail’s DNA percentage, the comet tail moment (TM) was determined.40 After completing all microscopic analyses, the data were decrypted.
Acknowledgments
The authors wish to thank the researchers in the Research Lab. of Molecular Carcinogenesis. Faculty of Science, Tanta University of Professor Elsayed. I. Salim for technical help. The authors declare that they received no funds throughout this work.
Author contributions
Eman El Nahass shared the experimental design, supervision, experimentations, data analysis, and interpretation and wrote the original manuscript. Safaa I. Abou Eldahab performed the experiments, data analysis, and statistics and shared in the writing of the original manuscript. Elsayed. I. Salim, conceptualization, experimental design, supervision, follow-up, data interpretation, and revised the final manuscript. All authors have read and approved the final manuscript.
Funding
This research did not obtain any specific financing from public, commercial, or nonprofit entities.
Conflicts of interest
The authors declare that they have no conflicts of interest.
