Introduction
For many years, cancer has affected many people and is known as a leading cause of death worldwide. This disease is considered a serious medical problem both in developed and less developed countries. The results of a survey based on the latest national cancer registration data in Iran predicted a 43% increase in the cases of cancer from 2017 to 2025 in Iran. Iran is located in a area with moderate risk of cancer based on the global cancer map. Pancreatic cancer is the most common type of cancer in the current century; it is one of the deadliest cancers related to the digestive system in the world. The most common treatment method for pancreatic cancer is surgery combined with chemotherapy.
Today, research on natural products has increased due to the increasing demand in the field of complementary medicine, which have low side effects and high safety. The treatment of diseases and the reduction of their symptoms by using herbal extracts has received more attention due to lower unwanted side effects and toxicity compared to synthetic compounds. Some of the compounds obtained from plants have even been used as models or precursors for making effective products with more effectiveness and less toxicity. Harmine is a beta-carboline alkaloid that is found naturally in many plants, but the main source of it is the pecan plant. It seems that the use of natural compounds obtained from plants along with the use of chemical drug can be more effective and useful. This study aims to assess the synergistic effect of fluorouracil combined with harmine on induction of apoptosis in AsPC-1 cell line and the expression of apoptotic genes (BAX, P53, Caspase-3, Caspase-9).
Methods
In this study, the AsPC-1 cell line was prepared from the cell bank of Pasteur Institute of Iran and cultured in RPMI1640 medium. When the density of cells reached 80%, the cytotoxicity of different concentrations of harmine and fluorouracil and their combination on human pancreatic cancer cells was evaluated by MTT method. For this purpose, the cells were cultured with a density of 2 x 104 cells in a 96-well plate. 24 hours later, the cells were treated with different concentrations of harmine and fluorouracil and their combination for 24 hours. After this period, cytotoxicity was evaluated. By using the DAPI staining method, it is possible to confirm the cytotoxic and antiproliferative effect of harmine, fluorouracil and their combination on ASPC-1 cells. First, 2 x 105 cells were cultured in each well of a 6-well plate for 24 hours in an incubator. Then the cells were exposed to harmine, fluorouracil, and their combination. It should be noted that the doses used in the DAPI test were determined based on the results of the MTT test and the IC50 calculated for the cells. After 24 hours, they were fixed with methanol for 10 minutes and then stained with DAPI for 10 minutes. Next, apoptotic nuclei were evaluated and photographed using a fluorescent microscope. Apoptosis was evaluated by annexin-propidium iodide diagnostic kit. In this regard, after 24 hours of culture in the plate, the cells were treated with harmine, fluorouracil and their combination for 24 hours. Then, the supernatant of the control group and the three treatment groups were transferred to Eppendorf and centrifuged at 4500 rpm for 8 minutes. Binding Buffer 1X, Annexin V-FITC and propidium iodide were added to the samples. Then, they were incubated at room temperature and in the dark, and its graphs were plotted using a flow cytometer. For gene expression assessment, 1x106 human pancreatic cancer cells were cultured for 24 hours after cell counting. Then, they were treated with harmine, fluorouracil, and their combination at specific concentrations for 24 hours. Next, RNA was extracted based on the protocol of the kit and the concentration was determined by the nanodrop method using a spectrophotometer. Afterwards, the extracted RNA was converted into cDNA. A pair of primers was designed by Oligo 7 software for each of the study genes and the internal control gene (Table 1).


Then, the amount of CG, temperature of the primer, and their specific connection were checked on the NCBI website and BLAST algorithm. Finally, the expression of apoptotic genes caspase-3, caspase-9, BAX and p53 was investigated using the SYBR green fluorescent stain with Real-Time PCR method in Bio Rad device.
3. Results
The results showed that, among the studied concentrations, 30 μg/ml harmin, 35 μg/ml fluorouracil, and the combination of 10 μg/ml fluorouracil and 20 μg/ml harmine caused the death of half of the cells (Figure 1). 

Morphological results showed that 24 hours after treatment with 30 μg/ml harmine, almost half of the cells left the polyhedral state and became spherical. In treatment with 35 μg/ml fluorouracil, the cells were rounded and out of the normal shape. This rounded shape was also observed in the combined group (20 μg/ml harmine + 10 μg/ml fluorouracil) (Figure 2). 

The results of DAPI staining test showed that in AsPC-1 cells, immediately after and 24 hours after treatment, the nuclei were fragmented and the shape of the nuclei was out of normal form and wrinkled, which indicates the occurrence of apoptosis in these cells. In the control group, the nuclei were healthy and intact (Figure 3). 

The results of annexin 5-propidium iodide test showed that, while 92% of the cells were alive in the control group, about 40% were alive and 52% were apoptotic in the harmine group with a concentration of 30 μg/ml. In the cells treated with 35 μg/ml fluorouracil, about 35% of the cells had undergone apoptosis. In the cells treated both harmine and fluorouracil, about 60% of the cells underwent apoptosis and only about 15% were alive (Figure 4 and 5).

Figure 4 shows the expression results of the study genes under the influence of harmine, fluorouracil and their combination compared to the control group. All four studied genes showed the significant synergistic effect of harmine and fluorouracil (p<0.05), which caused a significant increase in gene expression. The results related to the expression of caspase-3 and caspase-3 genes showed that although there was also an increase in the harmine and fluorouracil treatment groups, the increase in the synergistic group was only significant (p<0.05). In BAX and P53 genes, a significant increase in expression was seen only in the synergistic group; treatment with harmine and fluorouracil alone could not cause significant changes in gene expression.

Ethical Considerations
Compliance with ethical guidelines
All ethical principles were considered in this article. The participants were informed of the purpose of the research and its implementation stages. They were also assured about the confidentiality of their information and were free to leave the study whenever they wished, and if desired, the research results would be available to them.

Funding
This study was extracted from the master thesis (No. 162374163) of the first author. It was not funded by any organizations.

Authors' contributions
All authors equally contributed to preparing this article.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
The authors would like to thank the personnel of Research Institute in Kharazmi University for their cooperation.


References

1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA: A Cancer Journal for Clinicians. 2015; 65(2):87-108. [DOI:10.3322/caac.21262] [PMID]
2. Kanji ZS, Gallinger S. Diagnosis and management of pancreatic cancer. Canadian Medical Association Journal. 2013; 185(14):1219-26. [Link]
3. Salem AA, Mackenzie GG. Pancreatic cancer: A critical review of dietary risk. Nutrition Research. 2018; 52:1-13. [DOI:10.1016/j.nutres.2017.12.001] [PMID]
4. Sara JD, Kaur J, Khodadadi R, Rehman M, Lobo R, Chakrabarti S, et al. 5-fluorouracil and cardiotoxicity: A review. Therapeutic Advances in Medical Oncology. 2018; 10:1758835918780140. [DOI:10.1177/1758835918780140] [PMID] [PMCID]
5. Rouanet M, Lebrin M, Gross F, Bournet B, Cordelier P, Buscail L. Gene therapy for pancreatic cancer: Specificity, issues and hopes. International Journal of Molecular Sciences. 2017; 18(6):1231. [DOI:10.3390/ijms18061231] [PMID] [PMCID]
6. Shahbazfàr AA, Zare P, Mohammadpour H, Tayefi-Nasrabadi H, Emami SJ. Combination therapy enhanced the antitumor activity of artemisinin-iron in lung cancer Calu-6 cells. European Journal of Oncology. 2017; 22(1):31-7. [Link]
7. Cheng MH, Hsieh CL, Wang CY, Tsai CC, Kuo CC. Complementary therapy of traditional Chinese medicine for blood sugar control in a patient with type 1 diabetes. Complementary Therapies in Medicine. 2017; 30:10-13. [DOI:10.1016/j.ctim.2016.09.007] [PMID]
8. Ergin V, Hariry RE, Karasu C. Carbonyl stress in aging process: Role of vitamins and phytochemicals as redox regulators. Aging and Disease. 2013; 4(5):276-94. [DOI:10.14336/AD.2013.0400276] [PMID] [PMCID]
9. Javeed M, Rasul A, Hussain G, Jabeen F, Rasool B, Shafiq N, et al. Harmine and its derivatives: Biological activities and therapeutic potential in human diseases. Bangladesh Journal of Pharmacology. 2018; 13(3):203-13. [DOI:10.3329/bjp.v13i3.34990]
10. Balaji N, Devy AS, Sumathi MK, Vidyalakshmi S, Kumar GS, D’Silva S. Annexin v - affinity assay - apoptosis detection system in granular cell ameloblastoma. Journal of International Oral Health. 2013; 5(6):25-30. [PMID] [PMCID]
11. Gao J, Zhu H, Wan H, Zou X, Ma X, Gao G. Harmine suppresses the proliferation and migration of human ovarian cancer cells through inhibiting ERK/CREB pathway. Oncology Reports. 2017; 38(5):2927-34. [DOI:10.3892/or.2017.5952] [PMID]
12. M Milczarek M, Pogorzelska A, Wiktorska K. Synergistic interaction between 5-FU and an analog of sulforaphane-2-oxohexyl isothiocyanate-in an in vitro colon cancer model. Molecules. 2021; 26(10):3019. [DOI:10.3390/molecules26103019] [PMID] [PMCID]
13. Ding Y, He J, Huang J, Yu T, Shi X, Zhang T, et al. Harmine induces anticancer activity in breast cancer cells via targeting TAZ. International Journal of Oncology. 2019; 54(6):1995-2004. [DOI:10.3892/ijo.2019.4777]
14. Dun J, Chen X, Gao H, Zhang Y, Zhang H, Zhang Y. Resveratrol synergistically augments anti-tumor effect of 5-FU in vitro and in vivo by increasing S-phase arrest and tumor apoptosis. Experimental Biology and Medicine (Maywood). 2015; 240(12):1672-81. [DOI:10.1177/1535370215573396] [PMID] [PMCID]
15. Li Z, Chen L, He C, Han Y, Han M, Zhang Y, et al. Improving anti-tumor outcomes for colorectal cancer therapy through in situ thermosensitive gel loading harmine. American Journal of Translational Research. 2020; 12(5):1658-71. [PMID] [PMCID]
16. Gao J, Hou D, Hu P, Mao G. Curcumol increases the sensitivity of colon cancer to 5-FU by regulating Wnt/β-catenin signaling. Translational Cancer Research. 2021; 10(5):2437-50. [DOI:10.21037/tcr-21-689] [PMID] [PMCID]
17. Hamsa TP, Kuttan G. Harmine activates intrinsic and extrinsic pathways of apoptosis in B16F-10 melanoma. Chinese Medicine. 2011; 6(1):11 [DOI:10.1186/1749-8546-6-11] [PMID] [PMCID]
18. Abdel Latif Y, El-Bana M, Hussein J, El-Khayat Z, Farrag AR. Effects of resveratrol in combination with 5-fluorouracil on N-methylnitrosourea-induced colon cancer in rats. Comparative Clinical Pathology. 2019; 28(5):1351-62. [DOI:10.1007/s00580-019-02967-2]
19. Diao E, Ren D, Liu T, Zhang J, Hu W, Hou H. Ozone detoxification of patulin in aqueous solution and cytotoxic evaluation using human hepatic carcinoma cells. Toxicon. 2018; 155:21-26. [DOI:10.1016/j.toxicon.2018.10.004] [PMID]
20. Truter D, Chellan N, Strijdom H, Webster I, Rawstorne J, Kotzé SH. Histomorphological changes in the pancreas and kidney and histopathological changes in the liver in male Wistar rats on antiretroviral therapy and melatonin treatment. Acta Histochemica. 2018; 120(4):347-55. [DOI:10.1016/j.acthis.2018.03.006] [PMID]