Efficacy of Prunus armenica, Bee venom, and their combinations on p53 and Bcl-2 gene expression in human pancreatic and lung cancer cells

Malignancies are one of the world’s most serious health problems. Pancreatic and lung cancers are two of the most lethal types of cancer, with 5-year survival rates ranging from 10 to 20%. Despite decades of investigation, the molecular processes and selectivity of Prunus armenica and Bee venom as anticancer drugs are still largely un-understood. The goal of our study is to determine the expression levels ofp53 and Bcl-2 mRNAs obtained from human pancreatic (PANC-1) and lung (A549) cancer cells treated with Prunus armenica, Bee venom or their combinations by using MTT and real time PCR assays. We showed that in a concentration-dependent manner of Prunus armeniaca, bee venom, and a combination of both, the cell growth was significantly decreased. Our findings also revealed an adverse association between tumor suppressor (p53) and proto-oncogene (Bcl-2) gene expression in both cancer cells treated with diverse substances vs untreated. As a consequence, the tested compounds dramatically alter p53 and Bcl-2 gene expression, suggesting that they might be useful in the prevention or treatment of pancreatic and lung cancers.


Introduction
Cancer is the major cause of death globally, and it is defined as an uncontrolled and unregulated growth of cells capable of infecting other organs or tissues in the body [1].Pancreatic cancer (PC) is a deadly gastrointestinal illness with few effective treatments [2,3].It is the third greatest cause of cancer-related fatalities in the United States, with just 10% of patients living for yet more than five years [4,5].In Saudi Arabia, PC is one of the most lethal cancer with just a 10% 5-year survival rate [6].PC patients can be treated with surgery if the tumor is still confined, or with alternative therapies such as radiation, chemotherapy, chemoradiation, and targeted therapy if the tumor has metastasized, according to the National Cancer Institute.With a median survival of 2.5 years, operations may be conducted on less than 20% of all PC individuals (classified as acceptable and borderline respectable).The median survival duration for the remaining 80% of PC non-rejected individuals (classified as locally progressed and metastatic) is 3.5 months [7,8].Lung cancer is not in a better position than pancreatic cancer, which has been the most fatal illness in recent years.It is a highly aggressive, fast-expanding, and common malignancy.Lung cancer is categorized as non-small-cell lung cancer, which is the most common kind (80%), but accounts for just 15% of all diagnosed cases [9].Every year, almost two million individuals worldwide are diagnosed with lung cancer, with 90% dying [10].According to SEER, 52 percent of PC and 55 percent of lung cancer were diagnosed at the distant stage, which occurs when cancer cells infiltrate other organs [11].
For thousands of years, the honeybee has been the source of a variety of therapeutic commodities utilized by humans, including honey, propolis, and venom [12].The molecular mechanisms behind bee venom's anticancer effect, however, remain unknown, notably in pancreatic and lung malignancies, which are the major causes of cancer death in both males and females [13,14].Understanding the key mechanism and selectivity of bee venom against cancer cells is vital for designing and refining new effective medications derived from a natural resource that is abundant & affordable in several countries.Honeybee venom has been shown to have anti-tumor activity in lung cancer, leukemia, ovarian, cervical, and PC, with higher toxicity in transformed cells than in non-transformed cells [15][16][17][18].Apricot, on the other hand, has long been used to treat a number of diseases in traditional medicine due to its high quantities of amygdalin, vitamins C and K, -carotene, niacin, and thiamine [19,20].The anticancer effects of Prunus armeniaca extract have received little attention, prompting the current study.A growing amount of available data supports the anti-cancer properties of Prunus armeniaca extract in cancer therapy.
Many studies have been conducted in recent decades to better understand the molecular mechanism of carcinogenesis [21].The tumor suppressor (p53) and the proto-oncogene (Bcl-2) were two of the earliest cancer genes found [22].Apoptosis is a definite process that prevents cancer cell growth.A growing amount of evidence suggests that B-cell lymphoma-2 (Bcl-2) plays a role in apoptosis via p53 [23,24].As a crucial sensor of cellular stress, p53, the "guardian of the genome," plays a critical role in tumor suppression in humans.The primary idea behind p53 is to connect to the DNA and either repair the damage or lead the damaged cell to apoptosis.This is how p53 combats cancer cell aggregation [25,26].In transformed mouse cell lines, mutant p53 proteins were found, while Bcl-2 translocation was detected in lymphoma.Despite their shared tumor relevance, it was previously thought that they shared nothing else.Bcl-2 protein is required for directing the apoptosis process and has been associated with cell death suppression [27].Furthermore, elevated Bcl-2 expression was exclusively found in hematological tumors, but p53 mutations were common in solid tumors [28].We investigate changes in the expression patterns of the p53 and Bcl-2 genes after treating pancreatic cancer cell lines (PANC-1) and lung cancer cell lines (A549) with Prunus armeniaca extract, honeybee venom, or combinations of the two.

Tested compound preparation
Sigma Aldrich (St. Louis, MO, USA) and VACSERA (Dokki, Giza, Egypt) provided the Prunus armeniaca and Bee venom, respectively.The stock solutions were made at concentrations of 1000 μg / mill.The solutions were filtered, and the final concentrations in DMEM high glucose medium were provided.

Tissue culture
The PANC-1 cell line has been used as a model of human pancreatic cancer in this study and the A549 cell line as a model of human carcinoma non-small-cell lung cancer.Both cell lines were brought from the American Type Culture Collection, kindly provided by VACSERA Co., Egypt.The cells were grown in DMEM with 10% FBS and 1% antibiotic solution (Sigma-Aldrich, USA).Cells were grown in 5% CO 2 at 37 °C.When the cells achieved 85% confluence and were subculture, they were collected for 4 min with trypsin-EDTA for additional passages before being seeded and cultivated for 24 h prior to each test in all experiments.

MTT for cytotoxicity assay
This method was determined by using 3-(4,5-dimethylthiazol-2-yl)-2, 5-di-methyl tetrazolium bromide (MTT).To test the impact of Prunus armeniaca, Bee venom, and their mixtures on PANC-1 and A549 cancer cells, about 10 5 cells per well were seeded in a 96-well plate.After 24 h, serial dilution of each compound, namely 1000, 500, 250, 125, 62.5, 31.25,15.125, 7.8, 3.9, 1.9, and 1 μg/mL were added to the cells (untreated cells were used as a positive control and treated vero cell line were used as negative control).After incubation time, 20 μl of MTT (5 mg/ml) was added for 4 h at 37 °C.Followed by the removal of the solution and covering with tinfoil and stir cells for 15 min on an orbital shaker.The optical density was determined using a microplate reader (BMGLABTECH®FLUO star Omega, Germany) at 570 nm.Cell viability was calculated as a percentage of the mean optical density value obtained in comparison to controls, which was set at 100%.The 50% cytotoxicity (CC50) was identified by using the GraphPad PRISM program [29].The standard deviations were calculated for the fold of changes in gene expression after three repetitions.

cDNA synthesis
Total RNA was isolated according to the manufacturer's instructions using the QIAzol Lysis Reagent (Qiagen, Germany).The RNA purity and concentration were evaluated using a NanoDrop, and the RNA integrity was validated using electrophoresis.After that, 1 µg of RNA was utilized for cDNA synthesis with random primer by using the Rever-tAid cDNA Synthesis Kit (ThermoFisher, USA).

Real time PCR
The reaction was carried out using the primers listed in Table 1.The reaction was carried out in a final volume of 25 µl using SYBER Green Master Mix (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions.For amplification reactions, a Rotor gene 6000 Corbett detection system was utilized.The thermal cycling conditions were as follows: a 5-min activation stage at 95 °C, followed by 35 cycles of 94 °C for 15 s and 55 °C for 1 min [30].

Data analysis
The cycle threshold (CT) values were collected, where the CT value is defined as the cycle number necessary to cross the threshold.The ΔCt value was used to report mRNA expression.Relative expressions were calculated using the 2 −ΔΔCt method [16].The qRT-PCR assays were performed in triplicates and the data were presented as the mean ± standard error of the mean (SEM) where applicable.

Cytotoxicity of Prunus armeniaca on PANC-1 and A549 cancer cells
The cytotoxicity of Prunus armenica on two human cancer cells, PANC-1 and A549 was examined.Both cells were incubated for 72 h with varying concentrations of Prunus armenica.Cell viability was determined using MTT.Prunus armeniaca significantly reduced the growth and proliferation of PANC-1 cells in a concentration-dependent manner over the 72-h incubation period, as demonstrated in (Fig. 1a).Prunus armeniaca effectively suppressed PANC-1 cell growth at log doses (3.5-6), (P < 0.05).A549 cell viability was reduced at log concentrations of Prunus armenica (3.5-5.8)(P < 0.05), (Fig. 1b).The cells' IC50 values (mg/ml) were determined and reported in (Table 1).Cytotoxicity of Prunus armenica on two human cancer cells PANC-1 and A549 was conducted.

Cytotoxicity of Bee venom on PANC-1 and A549 cancer cells
The cytotoxicity of bee venom was tested on two human cancer cells, PANC-1 and A549.Both cells were incubated for 72 h with varying concentrations of Bee venom.Cell viability was determined using MTT.As shown in (Fig. 2a), bee venom significantly decreased the development and proliferation of PANC-1 cells in a concentrationdependent manner over the course of 72 h.At log doses (3-5), bee venom effectively reduced PANC-1 cell growth (P < 0.05).At log concentrations of Bee venom (3.5-5), cell viability of A549 was reduced (P < 0.05), (Fig. 2b).The cells' IC50 values (mg/ml) were determined and reported in (Table 1).

Cytotoxicity of mixed compounds on PANC-1 and A549 cancer cells
On two human cancer cells, PANC-1 and A549, the cytotoxicity of a composite IC50 of both drugs were tested.Both cells were treated with two-fold dilutions of the combined IC50 of both drugs for 72 h.Cell viability was determined using MTT.As demonstrated in (Fig. 3a), mixed substances notably reduced the growth and proliferation of PANC-1 cells in a concentration-dependent manner over the course of 72 h.At log doses (4-6), mixed substances effectively suppressed PANC-1 cell growth (P < 0.05).A549 cell viability was reduced at log concentrations of combined chemicals (4.5 -6), (P < 0.05), (Fig. 3b).The cells' IC50 values (mg/ ml) were determined and reported in (Table 1).

Prunus armeniaca, bee venom and their mixed effects on p53 in PANC-1 cells
The present study showed that the expression of the p53 gene in PANC-1 cells was extremely higher in the treated group than in the control group.Prunus armeniaca, Bee venom, and combined substances significantly boost p53 gene expression compared to the untreated group (P < 0.001), (Fig. 4a).

Prunus armeniaca, Bee venom and their mixed effects on Bcl-2 in PANC-1 cells
We found that the expression level of the Bcl-2 gene in PANC-1 cells was significantly lower in the treatment group compared to the control group.Prunus armeniaca, Bee venom, and combined chemicals substantially decreased the expression of the Bcl-2 gene in treated vs untreated groups (P < 0.001), (Fig. 4b).

Prunus armeniaca, Bee venom and their mixed effects on Bcl-2 in A549 cells
Our results found that the expression of the Bcl-2 gene in A549 cells was lower in the treated group than in the control group.As demonstrated in (Fig. 4c), Prunus armeniaca, Bee venom, and mixed compounds dramatically decreased Bcl-2 gene expression in treated groups vs untreated.

Prunus armeniaca, Bee venom and their mixed effects on p53 in A549 cells
This study found that the expression level of the p53 gene in A549 cells was significantly higher in the treated group compared to the untreated group.Prunus armeniaca, Bee venom, and combined chemicals substantially increased the expression of the p53 gene in treated vs untreated groups (P < 0.001), (Fig. 4d).

Discussion
In the recent decade, medicines used in cancer treatment have been produced from natural sources that include natural active components, such as Amygdalin, Prunus armeniaca, Ginseng, Honey, and Aloe Vera [31].The majority of cancer research has concentrated on developing a natural active substance capable of not only inhibiting carcinogenesis but also reversing promotional phases by triggering apoptosis and growth arrest in diverse cancer cells while having no harmful effects on normal cells [32].
Most cancers, according to recent reports, are caused by the failure of several genes that code for anti-apoptotic, growth factors, and tumor suppressors; these genes are potential cancer therapy targets [33].P53 and Bcl-2 genes have been linked to cancer formation because they control apoptosis and increase cell survival [34].
The current study focused on how Prunus armeniaca, bee venom, and their combination may limit the growth of A549 and PANC-1 cells in vitro.Furthermore, this work investigated the effects of Prunus armeniaca, bee venom, and their combination on the expression levels of the apoptotic regulating genes p53 and Bcl-2.Our findings clearly show that Prunus armeniaca, bee venom, and their combination have significant cytotoxic effects on lung cancer cells (A-549) and pancreatic cancer cells (PANC-1).Many studies have been conducted to investigate the cytotoxicity of Prunus armeniaca and bee venom against various malignancies, including liver carcinoma (HepG2), prostate carcinoma, and breast carcinoma cells (MCF-7) [16,17,35].The findings of this work lend credence to Prunus armeniaca and bee venom as potential preventive agents.
When we analyzed p53 and Bcl-2 genes expression under treatment with Prunus armeniaca, bee venom and their mixture, we found an adverse association between tumor suppressor (p53) and proto-oncogene (Bcl-2) gene expression in both cancer cells treated vs untreated.Realtime PCR analysis of gene expression levels revealed that Prunus armeniaca, bee venom, and their combination increased p53 expression while decreasing Bcl-2 expression in treated vs untreated pancreatic and lung cancer cells.Our results demonstrated the involvement of the intrinsic cell death pathway and suggest that the apoptosis induced by Prunus armeniaca and bee venom may be p53-dependent, as Bcl-2 expression is down-regulated in p53-dependent apoptosis, and thus the induction of p53-dependent apoptosis by Prunus armeniaca or bee venom has good potential for treating both cancers.These findings are consistent with those of a previous study, which discovered that increasing p53 and BAX expression while decreasing BCL-2 expression resulted in a significant increase in the Bax/Bcl-2 ratio, which is thought to be a driving force for apoptosis in human malignant melanoma and squamous carcinoma cells treated with tea tree oil [36].
P53 regulates the activity of Bcl-2 both directly and indirectly, as well as arresting cell development and activating apoptotic mechanisms that cause cell death [37,38].It may also suppress Bcl-2 activity by trans-activation of the cell division control protein 42 homolog (Cdc42), which starts a signaling cascade that leads to Bcl-2 phosphorylation and inactivation [39].Bcl-2 overexpression has been associated to acquire resistance to chemotherapy in malignancies according to Kirkin et al. [40].Changes in the expression of apoptosis-related genes might thus be employed as indications of their preventative effectiveness [36].In this study, Prunus armeniaca and bee venom elicited upregulation of P53 and downregulation of Bcl-2 after treatment in both cancer cells evaluated.Because of these effects, Prunus armeniaca and bee venom are promising preventative agents for pancreatic and lung malignancies.

Fig. 1
Fig. 1 Cytotoxicity effect of Prunus armenica on human pancreatic (PANC-1) and lung (A549) cancer cells.Both cells were treated with serial dilution of Prunus armenica for incubation time of 72 h. a Pru-

Fig. 2 Fig. 3
Fig. 2 Cytotoxicity effect of Bee Venom on human pancreatic (PANC-1) and lung (A549) cancer cells.Both cells were treated with serial dilution of Bee Venom for incubation time of 72 h. a Bee

Fig. 4
Fig.4 Evaluation of p53 and Bcl-2 relative genes expression levels under the effect of Prunus armenica, Bee venom using real-time PCR in A549 and PANC-1 cancer cells.a Expression of p53 on PANC-1 cell line was increased in the treated groups compare to the control group.b Expression of Bcl-2 on PANC-1 cell line was decreased in the treated groups compared to the control groups; c Expression of Bcl-2 on A549 cell line; Prunus armenica, Bee venom, and mixed compounds dramatically decreased Bcl-2 gene expression in treated groups; d Expression of p53 on A549 cell line; Prunus armenica, Bee venom, and mixed compounds substantially increased the expression of the p53 gene in treated VS untreated groups

Table 1
IC 50 values of PANC-1 and A549 cell lines incubated with Prunus armenicaand Bee venom for 72 h