Available online on 15.02.2024 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2024 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access  Full Text Article                                                                                                                                        Research Article

Gallic Acid and Doxorubicin Treatment HeLa cell lines by MTT assay

Serap Mutlu ÖZÇELİK OTÇU *, Murat AKKUŞ 

MD, Dicle University, Medical Faculty, Department of Histology and Embryology, Diyarbakır, Turkey

INTRODUCTION

Cervical cancer is an important health problem that threatens the lives of approximately 500,000 women worldwide every year. Smoking, exposure to human papillomavirus and immune system disorders are among the risk factors for cervical cancer. Although people with cervical cancer can be treated when the tumor is in its early stages, long-term morbidity from treatment is common.¹

Cervical cancer is one of the most common malignant tumors affecting women.² Cervical cancer is a significant cause of morbidity and mortality in women and is currently the second most common malignant disease in women worldwide.³ Increasing routine screening of the cervix plays a significant role in improving the disease prognosis by enabling the diagnosis of a large number of early-stage cervical cancer patients.⁴ In patients diagnosed with cervical cancer that has progressed to an inoperable stage or has recurred in other organs, commonly used anti-cancer chemotherapy, cisplatin-based chemotherapy drugs, is used.⁵ For this reason, there is a trend towards the discovery of new and more effective agents for patients with cervical cancer, as in many types of cancer.

Gallic acid is a trihydroxybenzoic acid that can be found in a variety of herbal medicines, foods, and beverages. A number of studies have demonstrated the potential anticancer activity of Gallic acid and its derivatives both in vivo and in vitro. In addition to its antitumor potential against cancer, it also plays a functional role in diabetes, hypercholesterolemia, cardiac hypertrophy, fibrosis and hypertension due to its excellent oxidation resistance.^6,7

Doxorubicin (Dox), sold under the trade name Adriamycin, is a chemotherapy drug. Doxorubicin is known as an inhibitor that prevents DNA synthesis by interfering with DNA strands. Although doxorubicin is toxic to both cancer and normal cells, the mechanism of cell death may not be similar in both cells.⁸ At the same time, Dox binds to the cell membrane and changes the physical properties of the membrane, thus impairing the membrane function of the cell.⁹

In this study we conducted with the HeLa cell line obtained from cervical cancer cells, the effects of Doxorubicin and Gallic acid in cell proliferation/migration and possible synergistic or antagonist effects were investigated.

MATERIAL AND METHODS

Cell Culture

10, 25, 50, 75, 100, 250, 500, 750 and 1000 µM/nM concentrations of GA/Dox applied to HeLa cell line and human skin keratinocyte cell line HaCat as healthy cell line were used. Dox and GA were obtained commercially and their concentrations were prepared in the appropriate solvent and applied to HeLa and HaCat cells.

MTT assay

In order to determine the IC50 doses in the groups where we will apply Doxorubicin and Gallic acid, HeLa and HaCaT cell lines were inoculated with the help of automatic multipipettes in 96-well culture dishes at a cell count of 3000-5000/well, respectively. After approximately 16 hours, serial dilutions were made in the dose range of doxorubicin 10-1000 nM and Gallic acid 10-1000 µM and incubated in plates with 9 different concentrations for 48, 72 h. When we analyzed cell viability in the MTT test, the outer wells of the culture dishes were excluded to reduce trial error. Each agent and vehicle control group was set to consist of 6 wells. MTT test was applied to analyze the cells remaining alive after incubation. According to the results of the MTT analysis, the effects of GA and Dox at different concentrations were calculated by using the SPSS 20 statistical package program and probit analysis for the IC50 values for each tumor cell line and chemotherapy agent in the control and experimental groups. 

Statistical analysis

In the study, the difference between the live cell ratios determined by the MTT test. In the comparison of the two groups, depending on the homogeneity of the data, the independed sample T test or the Mann Whitney U test were used. Analyzes were made with SPSS 20, (IBM, USA) program, and p ≤ 0.05 was used.

RESULTS

After HeLa and HaCat cells grown in T75 flasks became 90% confluent, they were treated with trypsin and removed and inoculated into 96-well culture dishes at 3000 cells/well to perform MTT analyses. In order to determine the IC50 doses of GA and Dox, 10-1000 µM amounts of GA and 10-1000 nM Dox were applied to these cultures and incubated for 48 hour. At the end of the incubation period, drugs were removed from the cultures by pipetting and spectrophotometric measurement was performed according to the ratio of tetrazolium salts formed by adding MTT solution. 48 hours of Dox application, the IC50 value was found to be 124.6 nM. Significant decreases in cell proliferation were observed as the dose increased. In the application that started with 100,000 cell cultivation, the average number of cells was obtained as 65,000 for all time periods in the Dox application at a concentration of 1000 nM. As a result of 48 and 72 hours of treatment, the number of cells were the lowest with 74.79 and 68.39. After the IC50 value was found as a result of the statistical analysis, it was determined that the cell viability decreased significantly after 25 nM concentration Dox application compared to the vehicle group (Table 1). 

Table 1: Dox application to HeLa cells for 48, 72 hours.

As a result of the data obtained by applying MTT test on HeLa cell series after GA application, % cell viability and IC50 value calculated using probit analysis and statistical data compared to the control are given in Table 2. On the other hand, a decreased cell viability was detected in the HaCaT cell line only as the GA dose increased. The survival rate of the HeLa cell line after GA application was found to be 70 on average compared to all times. The survival rate of the HaCaT cell line after GA application. Significant differences were detected between GA and HaCaT and HeLa cell lines. The IC50 value was obtained as 242.4 µM/L in the 48 h and 236.4 µM/L in the 72 h for GA application (Table 2). 

Table 2: GA application to HeLa cells for 48, 72 hours.

Dox and GA were also applied to the healthy cell series and their cytotoxic effects were analyzed. After the application of both agents, the % cell viability resulting from the MTT test in the HaCaT cell series and the IC50 values calculated using probit analysis and the statistical analyzes obtained when compared to the control are given in Table 3 and 4. IC50 value could not be obtained by applying Dox to the HaCaT cell line for 48, 72 hours. When compared to the vehicle control group, it was determined that significance was achieved after 48, 72 hours of Dox application and 50 nM concentration.

Table 3: Dox application to HaCaT cells for 48, 72 hours.

As a result of GA application, it was observed that there was a decrease in HaCaT cells depending on the dose increase. In the application that started with 100,000 cell cultivation, the number of cells was found to be 42.81 in the 48-hour GA application at a concentration of 750 µM and 7 in the 72-hour application (Table 4). As a result of statistical analysis, it was determined that cell viability decreased significantly after 500 µM GA application (Table 4).

Table 4: GA application to HaCaT cells for 48, 72 hours.

DISCUSSION

This study also showed that Gallic acid and Doxorubicin significantly suppressed the proliferation of HeLa cells and accelerated their apoptotic processes, which is consistent with the results of previous studies. Previous studies have revealed that GA inhibits cell proliferation and invasion of different tumors such as small cell lung cancer.¹⁰. It has played an important role especially in the treatment of breast and colon cancer. The occurrence of colon cancer is closely associated with the abnormal expression of multiple genes. Therefore, regulating the expression of certain key genes such as SRC during tumorigenesis to inhibit malignant transformation is an effective tool to control tumor growth. Although chemotherapeutic drugs are very effective, serious side effects and the development of drug resistance limit the use of these drugs. The use of natural products with anticancer activity may help partially overcome these problems. A number of studies investigated the cytotoxic effect of GA in various cell lines and showed that IC50 for Calu-6, A 549 and HeLa.¹¹ These results indicate that Gallic acid induces cell death in tumor cells with relatively high selectivity. In our study, it was observed that gallic acid treatment decreased the growth of HeLa cells in a dose-dependent manner with the IC50 value in 48 hours. The strong effect of gallic acid treatment seen in our study is similar to that reported by Sánchez-Carranza et al. ¹² They reported that gallic acid enhanced the cytotoxic effect of Paclitaxel in A2780 cells and A2780AD cells through treatment. They argued that the inhibition of proliferation and the arrest of the G2/M phase might be due to the ROS-mediated inhibition of kinases regulated by excessive ROS production by Gallic acid and by extracellular signals triggered by Paclitaxel.¹²

Chemotherapeutic drugs have a very important place in cancer treatment and although they are very effective in treatment, the use of these drugs is limited due to their side effects such as damage to vital organs such as the heart and liver, and the emergence of drug resistance over time. For this reason, every natural product with anticancer activity is the focus of attention and it is thought that it can help to overcome these problems, albeit partially. In this study, the ability of Gallic acid to be an alternative to chemotherapeutic agents such as doxorubicin in human HeLa cells.

Natural compounds such as flavonoids are recognized as important agents for cancer prevention and treatment because of their potential therapeutic effects and limited toxicity to healthy cells. In carcinogenesis, flavonoids interfere with intracellular signal transduction pathways, suppress proliferation, angiogenesis, metastasis, and increase apoptosis.^13,14 In this study, GA and DOX treatment decreased the growth of HeLa cells. The cytotoxic effect of GA can be explained by its pro-oxidant property, which is accepted as an apoptosis inducer in different cancer cell lines, especially HeLa cells.^15,16 The different IC50 values of GA on HeLa cell in our study can be explained by the fact that although MTT assay has been used in cancer research for 30 years,¹⁵ it rarely gives a consistent IC50 value for a particular chemical. He et al. attributed this problem to differences between manufacturers and to formulas used by different laboratories.¹⁷

Searches continue in different sources for the effects of substances obtained from natural sources such as Gallic acid. Moss and marine algae are popular targets. Studies have shown that subjects given algae and caraway extracts increase dregeneration in vital organs such as liver, kidney and pancreas.^18,19. In an in vitro study using gallic acid, it was reported thatcell viability decreased in cervical cancer cells.²⁰ It was alsoreported that natural antioxidants inhibited cell proliferation indifferent cancer cell lines (ovarian, breast, prostate, stomach, colon, nerve). In our gallic acid study, findings that will support the studies were obtained. Its effective role on the signaling mechanisms of both cell viability and proliferation has been demonstrated.^20-24 

CONCLUSİON

As a result, the cytotoxic effect of GA in cervical cancer was determined by the cell viability analysis test MTT. The findings were parallel to the anticancer findings of GA. In the HeLa cell line, the combination with the chemotherapy agent Dox caused cell death, and in addition to the antioxidant effect, it produced positive results. Also such as GA alternative antioxidants have a number of advantages such as lower dose and fewer advers effects. It is important to bring natural substances with antioxidant and anticancer activity, such as GA, to the clinic.

Acknowledgement: This study was a part of doctorate thesis of Serap Mutlu ÖZÇELİK OTÇU

Conflict of interest: No potential conflict of interest was reported by the author(s).

Ethic: Ethical approval is not required because commercially available cell lines are used as an in vitro study.

Financial Support: This study was founded by Dicle University Department of Scientific Research Projects (DÜBAP) with Project number (funding no: TIP. 23.027)

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