Synergistic Effects of 5-Fluorouracil, N-Acetylcysteine, and L-Arginine on Inhibition of Breast Adenocarcinoma Cell Invasion and Migration: In Vitro

Souvik Paul,

doi:10.5281/zenodo.14860913

Research Paper | Open Access
Volume 02 | Issue 02 | Article Id IJPS/240210037

Synergistic Effects of 5-Fluorouracil, N-Acetylcysteine, and L-Arginine on Inhibition of Breast Adenocarcinoma Cell Invasion and Migration: In Vitro
Souvik Paul*
Guru Nanak Institute of Pharmaceutical Science and Technology, Kolkata, West Bengal 700114, India

Abstract

The study sought to find out the effectiveness of combining 5-fluorouracil with N-Acetyl Cysteine and L-arginine in reducing the invasive and migratory properties of breast cancer cells. The Sorensen method was used to make a phosphate buffer solution, which was then evaluated for the haemolytic activity of NAC, 5-FU, and L-arginine on red blood cells. The effects of these drugs on breast cancer cells were subsequently investigated in vitro, with cell viability measured using the MTT assay. Cell lysates were collected by the freeze-thaw technique. The combination of 5-fluorouracil, N-Acetyl Cysteine, and L-arginine dramatically reduced breast cancer cell invasion and migration as compared with individual treatments or control groups. NAC's high antioxidant qualities effectively reduced breast cancer cell migration and invasion, whereas L-arginine had anti-cancer characteristics. As doses of drugs increased, the expression of Vascular Endothelial Growth Factor and Matrix Metalloproteinase-09, both linked with cancer growth, decreased significantly. When coupled with NAC and L-arginine, 5-FU further reduced VEGF and MMP-9 expression. The MTT test revealed that increasing the dosage of the drug combination resulted in slower cell growth. The therapeutic combination of NAC, L-arginine, and 5-FU shown significant effectiveness in inhibiting breast cancer cell invasion and migration. The results show that the combination of these drugs greatly lowers VEGF and MMP-9 expression, which contributes to the suppression of cancer cell growth and metastasis. The study focusses on NAC, L-arginine, and 5-FU as a possible therapeutic method for breast cancer therapy.

Keywords

Breast Adenocarcinoma Cells (BAC), 5-Fluorouracil (5-FU), N-Acetyl Cysteine (NAC), L-Arginine, Invasion and Migration Inhibition, VEGF and MMP-09 Expression

Introduction

Ethnopharmacology brings together conventional healers, botanists, anthropologists, chemists, and pharmacologists to research the conventional use of medicinal plants and create novel medications. However, despite their experience in clinical practice and contributions to the creation of contemporary medications derived from herbal sources, researchers have been mostly absent from this area of study^[1,2]. The cost of developing new drugs has gone up as a result of this lack of participation. Researchers can play a significant role in ethnopharmacological research to address this issue^[3,4]. Researchers can contribute by spending time in the field, addressing traditional practitioners, and learning about the ailments that herbal treatments are used to cure^[5]. In the context of contemporary drugs, they can also read previous works to suggest the proper application of conventional therapies. Researchers can direct lab studies on plants found during field study by working with pharmacologists. Additionally, collaborating with local healers can support the development of indigenous healthcare systems^[6,7].

Involvement in ethnopharmacological research can improve natural product-based drug development, provide more accurate information on conventional medicinal plant applications, and bridge the gap between conventional and contemporary medicine^[8]. The creation of drugs may be more targeted and less expensive as a result of the fusion of conventional knowledge with innovative scientific methodologies^[9]. In Ayurvedic medicine, the idea of "Designer Medicine" (DM) enables the creation of customised remedies depending on a patient's constitution and the properties of the materials utilized^[10]. These customised drugs can be prescribed and administered by ayurvedic practitioners as well as by Chinese and homoeopathic medical professionals. Ayurvedic medical concepts may be used to develop DMs, and the right software can help match therapeutic characteristics with patient demands^[11–14].

Antioxidants are crucial in the fight against cancer. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), two types of free radicals, can lead to oxidative stress and harm cells^[15–17]. Fruits, vegetables, and other naturally occurring sources of antioxidants help scavenge free radicals and guard against oxidative stress-related disorders, such as cancer. Finding naturally occurring antioxidants, especially those with plant origins, is of great importance^[18,19]. Breast cancer is a form of cancer that typically affects women but can also, although less frequently, affect males^[20,21]. Breast cancer officially starts in the cells of the breasts. It is one of the most common diseases in the world and the main reason why women suffer from cancer^[22–24]. This research looked into the effect of 5-fluorouracil (5-FU) when used with N-acetylcysteine (NAC) and L-arginine on BAC to learn more about the possible therapeutic advantages of combining these drugs in the treatment of breast cancer^[25]. 5-FU is a chemotherapeutic drug that is extensively used to treat different tumours; however, its efficacy can be restricted^[26]. NAC is an antioxidant that has been researched for its possible anti-toxicity and anti-oxidative stress properties. L-arginine is an amino acid that aids in cellular activities such as immune function and wound repair^[27,28]. The goal of this evaluation is to determine if the combination of 5-FU with NAC and L-arginine can enhance the anti-cancer effects of 5-FU and inhibit breast adenocarcinoma cell invasion and migration. The findings from such studies can provide valuable insights into potential combination therapies for breast cancer treatment and may contribute to the development of more effective treatment strategies.

MATERIAL & METHODS

Phosphate Buffer Solution (PBS) By Sorensen Method:

The method comprises making a solution of NaCl (Sodium Chloride), KH2PO4 (Potassium Dihydrogen Phosphate), and Sodium Hydrogen Phosphate (Na2HPO4) in distilled water. All of the substances were weighed, put to an autoclaved beaker, and stirred with a magnetic stirrer to dissolve them.

The pH of the mixture is then reduced to 7.2 by the addition of NaOH (Sodium hydroxide). The pH had been modified since it altered the buffer's capacity and the stability of the solution. The buffer solution was then autoclaved and filtered using filter paper to remove the impurities^[29].

Preparation Of Drug Solution For Haemolytic Activity: Drug solutions of NAC, 5-FU, L-Arginine were made after weighing 1ug in digital balance and dissolved in 1ml PBS.

Haemolytic activity using NAC, 5-FU and L-arginine:

Assessment of haemolytic activity of L-arginine, N-acetylcysteine, and 5-fluorouracil initiated with the collection of blood samples, which were then centrifuged to separate the Red Blood Cells (RBC) from the supernatant. Different concentrations of the drug were added to the tubes containing RBC and PBS after the relevant substance was produced as a stock solution in PBS. The IC50 value, which denotes the concentration at which 50% of the RBC was lysed, was determined after the tubes were incubated and centrifuged once more. These studies provided significant information on the effects of 5-FU, NAC, and L-arginine on blood cells by evaluating the possible haemolytic effects of these substances on RBC^[30].

NAC

Table 1: Haemolytic activity of NAC

Sample No	NAC Conc. (µg/ml)	PBS (µl)	RBC (µl)	Total(µl)
1	0	500	500	1000
2	10	490
3	25	475
4	50	450
5	100	400
6	D.W (500 µl)
7	H2O2 (10 µl)	490

5-FU

Table 2: Haemolytic activity of L-Arginine

Sample No	5-FU Conc. (µg/ml)	PBS (µl)	RBC (µl)	Total (µl)
1	0	500	500	1000
2	10	490
3	25	475
4	50	450
5	100	400
6	D.W (500 µl)
7	H?O? (10 µl)	490

L-Arginin

Table 3: Haemolytic activity of L-Arginine

Sample No	L- Arginine Conc. (µg/ml)	PBS (µl)	RBC (µl)	Total (µl)
1	0	500	500	1000
2	10	490
3	25	475
4	50	450
5	100	400
6	D.W(500 µl)
7	H2O2(10µ)	490

In vitro study of NAC, 5-FU and L-arginine on breast adenocarcinoma cells: Breast adenocarcinoma cells (BAC) were obtained from the Chittaranjan National Cancer Research Institute in Kolkata and maintained in the laboratory. A media solution was prepared using 90% Roswell Park Memorial Institute Medium (RPMI) and 10% foetal bovine serum (FBS), which was then filtered using membrane filtration.

NAC: Five Eppendorf tubes were labelled and filled with varying concentrations of N-acetylcysteine (NAC) ranging from 0 to 100 g/ml. AC cells and medium were added to each tube to reach a total volume of 1.5 ml.

Table 4: Haemolytic activity of NAC

SL No.	Conc. of Drug (µg/ml)	Drug (NAC) (µl)	Cell (µl) (2 x 10⁶)	Media (RPMI+10?S) (µl)	Total Volume(µl)
1	0	0	30	1470	1500
2	10	15	30	1455
3	25	37.5	30	1432.5
4	50	75	30	1395
5	100	150	30	1320

5-FU: Five labelled Eppendorf tubes received a fixed concentration of 10 g/ml of 5-fluorouracil (5-FU), along with BAC cells and medium

Table 5: Haemolytic activity of 5-FU

SL No.	5-FU (µl)	Cell (µl)(2x10⁶)	Media (RPMI+10?S) (µl)	Total Volume(µl)
1	10	30	1460	1500
2	10	30	1460
3	10	30	1460
4	10	30	1460
5	10	30	1460

L-Arginine: Five Eppendorf labelled tubes filled with different concentrations of L-arginine (0, 10, 25, 50, and 100 g/ml). BAC cells and medium were added to each tube.

Table 6: Haemolytic activity of L-arginine

SL No.	Conc. of L-Arginine (µg/ml)	Cell (µl) (2 x 10⁶)	Media (RPMI+10?S) (µl)	Total Volume(µl)
1	0	30	1470	1500
2	15		1455
3	37.5		1432.5
4	75		1395
5	150		1320

All tubes were incubated for 24 hours, followed by the MTT assay and freeze-thaw method to prepare cell lysates.

Freeze thaw method for cell lysate preparation

5-FU+ NAC

Table 7: Diff conc. of 5-FU and NAC on breast adenocarcinoma cells (BAC) for In vitro study

SL No.	Conc. of Drug (µg/ml)	Drug (NAC) (µl)	5FU (µl)	Cell (µl) (2 x 10⁶)	Media (RPMI+10?S) (µl)	Total Volume(µl)
1	0	0	10	30	1470	1500
2	10	15	10	30	1445
3	25	37.5	10	30	1422.5
4	50	75	10	30	1385
5	100	150	10	30	1310

5-FU+ L-arginine

Table 8: Diff conc. of 5-FU and L-arginine on breast adenocarcinoma cells (BAC) for In vitro study

SL No.	Conc. Of Drug (µg/ml)	Drug (L- arginine) (µl)	5FU (µl)	Cell (µl) (2x10⁶)	Media (RPMI+10?S) (µl)	Total Volume(µl)
1	0	0	10	30	1470	1500
2	10	15	10	30	1445
3	25	37.5	10	30	1422.5
4	50	75	10	30	1385
5	100	150	10	30	1310

The precipitated cells and supernatant were obtained after centrifuging the BAC cells (NAC and L-arginine in conjunction with 5-FU) for 24 hours. For cell lysis, 1% SDS was added to the tubes containing the cells, and the freeze-thaw method was repeated three times. After centrifugation, the cell debris precipitated, but the supernatant containing protein from the cells was recovered. Repeating the process facilitated the collection of more supernatant in Eppendorf tubes for later use^[31].

MTT Assay For Determining Cell Viability: Cell viability or proliferation was determined using the MTT assay. MTT powder was dissolved in PBS and added to Eppendorf tubes containing BAC cell supernatant, NAC & L-arginine, and medium. After 2 hours of incubation in a CO2 incubator, DMSO is added, followed by another hour of incubation. Finally, the absorbance was measured at 546 nm using a UV-VIS spectrophotometer. It helped in determining the effectiveness of NAC & L-arginine against breast cancer cells^[32,33].

RESULTS

Haemolytic Activity of 5-FU

Fig 1: Haemolysis % vs 5-FU conc. The dose which is shown in the graph indicates that it inhibits 50% of cell growth. 1% RBC was maintained in PBS then it was treated with different conc. of 5-FU. Haemolytic activity was checked by UV-vis spectroscopy after 1hr incubation.

Graphical Representation of Estimation of VEGF of 5-FU, NAC and L-Arginine

Fig 2: NAC and L-Arginine was used with and without 5-FU. After collection of BAC, they were treated with NAC/L-Arginine and incubated for 24 hr. SDS was added after centrifugation and freeze thaw method was done for cell lysis to obtain the VEGF. Individual experiments are expressed as Mean+Sem. All data were analysed using one-way ANOVA. Differences were considered significant at *p<0>.

MMP-09 of 5-FU, NAC and L-Arginine

Fig 3: NAC and L-Arginine was used with and without 5-FU. After collection of BAC, they were treated with NAC/L-Arginine and incubated for 24 hr. SDS was added after centrifugation and freeze thaw method was done for cell lysis to obtain the MMP-09. Individual experiments are expressed as Mean+Sem. All data were analysed using one-way ANOVA. Differences were considered significant at *p<0>.

MTT Assay of 5-FU, NAC and L-Arginin

Fig 4: Cell viability % vs Conc. of Drugs. NAC and L-Arginine was used with and without 5-FU. After collection of BAC, they were treated with NAC/L-Arginine and incubated for 24 hr cell was tested for MTT assay. Individual experiments are expressed as Mean+Sem. All data were analysed using one-way ANOVA.

DISCUSSIONS:

Finding effective therapies for breast cancer is quite challenging. Study of the possible synergistic effects of 5-Fluorouracil (5-FU) in conjunction with N-Acetylcysteine (NAC) and L-Arginine helps to optimize therapy regimens for better outcomes for patients. It is crucial to prevent invasion and migration in order to avoid metastasis, which has become a major contributor to cancer-related fatalities^[34]. The potential of this combination therapy to suppress metastasis, hence potentially limiting the spread of cancer cells, is determined by evaluating its effects on breast adenocarcinoma cell behavior in vitro. Due to their ability to target cancer cells via a variety of pathways, lower drug resistance, and increased therapeutic efficacy, combination treatments have become more significant^[35,36]. The study of 5-FU with NAC and L-Arginine advances our understanding of combination treatments and opens the door to the investigation of cutting-edge therapeutic approaches. The development of personalized medicine is facilitated by an understanding of the effects of various combinations in vitro, enabling customized treatment regimens based on unique patient features. In vitro studies are also important preclinical evaluations, offering preliminary information on effectiveness and safety profiles that can direct subsequent research in animal models and clinical trials and perhaps result in the creation of novel treatment approaches. Results from the graphs demonstrate how BAC cells invade, migrate, and proliferate at different medication dosages. Initially, RBC cells were exposed to in vitro haemolytic activity to assess the safety profile of the drugs. 5-FU concentration and haemolysis % are compared in Fig. 1. The 50% inhibition of cell growth indicated by the IC50 value. Before being treated with various doses of 5-FU, 1% RBC was maintained in PBS. UV-vis spectroscopy was used to measure the haemolytic activity after one hour of incubation. The IC50 values for NAC and L-Arginine were also identified, showing that only very low concentrations of drugs are required to 50% restrict cell growth. As a result, it can be inferred from all three IC50 graphs that 5-FU, NAC, and L-arginine are safe medications. Based on the estimation of VEGF of 5-FU, NAC, and L-arginine, Fig. 2 displays the VEGF level observed for various concentrations. NAC and L-arginine combined vs. VEGF ng/ml. Both 5-FU and NAC were utilized, as well as L-arginine. They had NAC/L-arginine therapy after BAC collection, followed by a 24-hour incubation period. SDS was added after centrifugation and freeze thaw method was performed to estimate the VEGF. Individual tests are presented as Mean+Sem. The entire set of data was analyzed using one-way ANOVA. Differences were considered significant at *p<0> In the first plot, NAC concentration is plotted against VEGF level, and it can be observed that as the drug's concentration rises, VEGF levels fall. The same pattern is seen when L-arginine concentration is compared to VEGF level. So, based on the graph, we can conclude that NAC is a strong antioxidant. Both NAC and L-arginine have demonstrated a decrease in VEGF expression with an increase in their concentration when combined with 5-FU. So, L-arginine worked effectively and NAC was an effective antioxidant.

Fig 3 shows the MMP-09 level was detected for different drug concentrations based on the graphical depiction of calculation of MMP-09 of 5-FU, NAC, and L-arginine. MMP-09 ng/ml vs. NAC and L-arginine conc. L-arginine and NAC were administered in conjunction with and without 5-FU. Following BAC collection, they were treated with NAC/L-arginine and incubated for 24 hours. To get MMP-09, SDS was added after centrifugation and cell lysis was performed using the freeze-thaw technique. Individual experiments are denoted by the Mean+Sem. One-way ANOVA was used to analyse all of the data. At *p<0>

CONCLUSIONS:

The study discovered that N-Acetyl Cysteine (NAC) has high antioxidant effects and successfully prevented the invasion and migration of breast cancer cells. L-Arginine, on the other hand, displayed anti-cancer properties on these cells. The expression of VEGF and MMP-09, which are linked to cancer growth, decreased dramatically as the drugs concentration rose. With increasing concentrations of 5-FU, an antimetabolite/anticancer drug, cancer cell proliferation was successfully reduced. When coupled with 5-FU, NAC and L-Arginine both demonstrated a reduction in VEGF and MMP-09 expression as their concentrations emerged. The MTT Assay, which was used to measure cell viability, demonstrated a reduction in cell growth as the drug concentration rose. Overall, these data indicate that NAC, L-Arginine, and 5-FU have potential as therapeutic agents against breast adenocarcinoma cells.

CONFLICT OF INTERESTS:

The author declares no conflict of interests.

FUNDING:

The author did not receive any financial support, funds or grants from any organization for the submitted work.

ACKNOWLEDGEMENTS:

The author would like to thank Department of Science and Technology (DST-Govt. of India), Guru Nanak Institute of Pharmaceutical Science and Technology, Kolkata, India, for providing support and facilities to conduct this research work.

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Reference

Grabley S, Thiericke R. Bioactive agents from natural sources: trends in discovery and application. Adv Biochem Eng Biotechnol. 1999;64:101–54.
Kim JS, Roberts JM, Bingman WE, Shao L, Wang J, Ittmann MM, et al. The prostate cancer TMPRSS2:ERG fusion synergizes with the vitamin D receptor (VDR) to induce CYP24A1 expression-limiting VDR signaling. Endocrinology (United States). 2014 Sep 1;155(9):3262–73.
Raza M. A role for physicians in ethnopharmacology and drug discovery. J Ethnopharmacol. 2006 Apr 6;104(3):297–301.
Liu GG, Eggleston K, Hu TW. Emerging Health Economics and Outcomes Research in the Asia-Pacific Region. 2008;
Ningombam DS, Devi SP, Singh PK, Pinokiyo A, Thongam B. Documentation and Assessment on Knowledge of Ethno-Medicinal Practitioners: A Case Study on Local Meetei Healers of Manipur. IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS [Internet]. 2014 [cited 2023 May 19];9(1):53–70. Available from: www.iosrjournals.orgwww.iosrjournals.org53|
Suroowan S, Mahomoodally MF. A comparative ethnopharmacological analysis of traditional medicine used against respiratory tract diseases in Mauritius. J Ethnopharmacol. 2016 Jan 11;177:61–80.
Baber NS, Ritter JM, Aronson JK. Medicines regulation and clinical pharmacology. Br J Clin Pharmacol [Internet]. 2004 Dec 1 [cited 2023 May 19];58(6):569–70. Available from: https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-2125.2004.02341.x
Jiang M, Zhao S, Yang S, Lin X, He X, Wei X, et al. An “essential herbal medicine”—licorice: A review of phytochemicals and its effects in combination preparations. J Ethnopharmacol. 2020 Mar 1;249:112439.
Jakka S, Rossbach M. An economic perspective on personalized medicine. HUGO Journal [Internet]. 2013 Apr 19 [cited 2023 May 19];7(1):1–6. Available from: https://thehugojournal.springeropen.com/articles/10.1186/1877-6566-7-1
Fu S, Stojanovska N. Designer Drugs. Encyclopedia of Forensic Sciences: Second Edition. 2013;36–44.
Devasagayam TPA. Introduction to Serial Reviews: Recent Advances in Indian Herbal Drug Research. J Clin Biochem Nutr. 2007 Mar;40(2):73–73.
Sontheimer H. “Neuro”-dictionary. Dis Nerv Syst. 2015;467–500.
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Souvik Paul

Corresponding author

Guru Nanak Institute of Pharmaceutical Science and Technology, Kolkata, West Bengal 700114, India

Souvik Paul*, Synergistic Effects of 5-Fluorouracil, N-Acetylcysteine, and L-Arginine on Inhibition of Breast Adenocarcinoma Cell Invasion and Migration: In Vitro, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 2, 951-961. https://doi.org/10.5281/zenodo.14860913

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Synergistic Effects of 5-Fluorouracil, N-Acetylcysteine, and L-Arginine on Inhibition of Breast Adenocarcinoma Cell Invasion and Migration: In Vitro

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Souvik Paul

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