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ORIGINAL ARTICLE
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Chlorogenic acid inhibits growth of 4T1 breast cancer cells through involvement in Bax/Bcl2 pathway


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom, Iran

Date of Submission10-Apr-2019
Date of Decision19-May-2019
Date of Acceptance12-Sep-2019
Date of Web Publication07-Jul-2020

Correspondence Address:
Azam Moslehi,
Cellular and Molecular Research Center, Qom University of Medical Sciences, Qom
Iran
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_245_19

 > Abstract 


Objective(s): Chlorogenic acid is an herbal compound with various effects such as antiviral, antioxidant, and anticancer effect with low toxicity, which inhibits cell proliferation. Clinical studies had shown that chlorogenic acid has a positive effect on the different types of cancers treatment. Hence, this study evaluates chlorogenic acid effects on 4T1 breast cancer cells.
Materials and Methods: In this study, cell proliferation was measured using an 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay (MTT) on 4T1 cells. Afterwards, other assays like P53, Caspase-3 proteins expression and Annexin V/PI were detected by flow cytometry. Also; Bax and Bcl-2 were carried out by immunocytochemistry.
Results: 200 μM of chlorogenic acid concentration showed the highest level of cytotoxicity toward 4T1 cells. Percentage of cell viability data were significant in 100 μM (P < 0.05) and 150, 200 μM (P < 0.001) doses. The evaluation using Annexin V/PI showed cell apoptosis in 100 μM (P < 0.05), 150 μM (P < 0.01), and for 200 μM (P < 0.05 and P < 0.01). Immunocytochemistry results showed the upregulation of Bax and also the downregulation of Bcl-2 in 4T1 cells treated with chlorogenic acid (P < 0.001). The expression level of P53 and caspase-3 increased during treatment with chlorogenic acid in the 4T1 cells (P < 0.001).
Conclusion: Our findings demonstrated that chlorogenic acid plays a notable role on apoptosis inducing in the 4T1 cells through regulation of apoptotic proteins.

Keywords: 4T1cell, apoptosis, Bax, Bcl-2, caspase-3, chlorogenic acid, P53



How to cite this URL:
Changizi Z, Moslehi A, Rohani AH, Eidi A. Chlorogenic acid inhibits growth of 4T1 breast cancer cells through involvement in Bax/Bcl2 pathway. J Can Res Ther [Epub ahead of print] [cited 2020 Oct 28]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=289131




 > Introduction Top


Breast cancer is one of the most prevalent kinds of women's cancer in all over the world.[1] Nowadays, there is a major requirement for detecting new treatments for breast cancer.[2] Apoptosis is a type of cell death in multicellular organisms, which its adjustment is necessary for natural growth, homeostasis, development, and cancer therapy. Changes in normal inducing to the apoptosis can lead to provide unusual cells formation, uncontrolled cell division, and reposition of mutations. Therefore, the adjustment of apoptosis is an important point in the cancer therapy.[3] Both Bax and Bcl-2 are members of the Bcl-2 family proteins, promote apoptosis, and relate to the mitochondrial function closely. When the Bax expression increases, it boosts apoptosis and vice versa. And when the Bcl-2 expression is high, apoptosis is blocked.[4] In addition, different studies have been performed on the role of Bax and Bcl-2 on various cancers; for example, colorectal,[5] lung,[6] breast,[7] melanoma,[8] and prostate cancers.[9] P53 tumor suppressor protein is an important regulator of Bax and Bcl-2 genes expression, which has several uses in DNA repair mechanisms, activation of cell cycle checkpoints, and apoptosis response for holding genomic consistency.[10] The mutant P53 misses its tumor inhibitor action, which is easy to find.[3] It plays a basic role in the cellular events, which happen after the DNA damage because of exposure to ultraviolet radiation. After DNA damage, the production of P53 in cells increases, and it leads to cell-cycle detention, DNA repair, and also apoptosis, if the damage is too large to be reformed. Mutations have been observed in the P53 gene in more than 50% of all cancers. The P53 disabling leads to mutations, and in the case that DNA damage is large, the cells become incapable of responding normally to cell cycle checkpoints or cell death undergoing programmed.[11] Various biochemical pathways exist during apoptosis, which eventually converge and would result to the activation of caspase families. Caspase pathways are known as appropriate nomination targets for anticancer's drug discovery.[12] Caspase response can aid cell cancer to become an apoptotic. Caspase-3 is one of the impressive caspases and can be measured to get the amount of apoptosis in cancer cells. Caspase-3 can be inactivated via external or internal factors.[13] In addition to that, recent studies demonstrated the roles of caspase-3 and P53 in inducing apoptosis of breast cancer.[14] Change of caspase-3 expression seems to be a factor, which helps the improper apoptosis in cancer cells.[15] Today, one of the main goals of different studies is to find new compounds for the cancer treatment, and many studies are focused on herbal based compounds with therapeutic effects.[16] Polyphenols are some kinds of compounds, which are abundant in plants. Polyphenols have inhibitory effects on cancer cell proliferation, angiogenesis, tumor growth, metastasis, inflammation, and inducing apoptosis as well. Moreover, they can protect normal cells against the free radicals lesion and adjust immune response system.[17] Chlorogenic acid is a polyphenol with a long history of usage in Chinese medicines.[18] It can be found in some plants such as honeysuckle, Cortex Eucommiae, Semen Coffea Arabica, and also green tea. So far, various reports about antioxidant, anti-inflammation,[19] antitumor, antiviral, eliminating free radicals, and anticancer effects of chlorogenic acid have been received.[20] Polyphenols have low toxicity and can inhibit cell proliferation. Several clinical studies have shown that treatment with chlorogenic acid affects the treatment of brain, lung, and colon cancers positively, as well as breast tumors and chronic myeloid leukemia.[21] However, there is still no report about chlorogenic acid effects on apoptosis induction in the 4T1 cells. 4T1 cells have a high growth rate and are extremely aggressive and metastatic. Therefore, the purpose of this research is to assess the apoptotic effects of chlorogenic acid on 4T1 mouse breast cancer cells.


 > Materials And Methods Top


Chlorogenic acid

To do this experimental study, chlorogenic acid (C16H18O9) was purchased from Sigma Aldrich Chemical Co. (C3878-1G) and dissolved in a sterile saline by stirring on a magnetic stirrer in the room temperature.

Preparation of sample solution

At first, a stock solution with sterile saline was prepared, and then, dilutions of 50, 100, 150, and 200 μM were made. Zero concentration was considered as the control group.

Cell culture

4T1 mouse breast cancer (ATCC® CRL2539™) cells were purchased from Pasteur Institute (Tehran, Iran). The cells were cultured in T75 flasks containing DMEM (BIO-IDEA, 12800-116) with 1% penicillin-streptomycin (BIO-IDEA, BI1036) and 10% FBS (BIO-IDEA, 10270-106). Next, the cells were cultured at 37°C, and 5% CO2 with 98% relative humidity, followed by washing with phosphate-buffered saline (PBS) 1X (BIO-IDEA, BI1038-500) at pH 7.4, and isolation by trypsin-EDTA 0.25% (BIO-IDEA, BI-2602). The grown cells were used during the experiments.

MTT cell proliferation assay

The half-maximal inhibitory concentration (IC50) of chlorogenic acid was specifically on 4T1 cells, line by MTT assay. 4T1 cells were seeded in 96 well tissue culture plates at a concentration of 2 × 104 cells/well. After that, the cells were glued to the surface at 37°C and 5% CO2 for 24 h. In the next day, concentrations of 50, 100, 150, and 200 μM of chlorogenic acid solution were exposed to the cells. After 24 h of incubation, washing was carried out with PBS, 10 μL of MTT solution was attached to each well, and the plates were incubated for 4 h. Subsequently, 100 μL of dimethyl sulfoxide was added, and the formazan crystals formed were dissolved. The cells incubated in alone medium culture were served as a control for cell viability. Cytotoxicity was calculated as the concentration of chlorogenic acid, which inhibited cell growth by 50% (IC50). All experiments were repeated at least for three times. By using an ELISA Reader (Bio-Tek), optical densities were read at 570 nm. Eventually, the percentage of cell viability was calculated according to the following formula: cell viability (%) = (A570 of treated cells/A570 of control cells)× 100.

Annexin V/PI staining

Apoptosis induction was measured using Annexin V-propidium iodide (PI) staining (FITC Annexin V, Biolegend, BI 85607), according to the manufacturer's protocol and flow cytometry (BD FACS Calibur) in the 4T1 cells. In summary, 105 of 4T1 cells were grown in 24 well plates and incubated for 24 h. After being treated with 50, 100, 150, 200 μM doses and incubated for 48 h, the cells were washed with cold phosphate buffer solution, and the contents of each well were centrifuged, separately. Then, the superficial fluid was discarded, and also, FITC Annexin V and PI solution (Biolegend, BI80508) were added. Afterward, the cells were gently vortexed and incubated for 15 min in darkness and at room temperature (25°C). Finally, Annexin V-binding buffer was added to each tube, and their contents were then analyzed by flow cytometry.

Bax and Bcl-2 expression by immunocytochemistry

About 2 × 105 of cells were poured into each one of the 24 well tissue culture plates and incubated for 24 h. After adding chlorogenic acid solution (200 μM), incubation was carried out for 48 h. 4% paraformaldehyde was added to fix the cells, and the contents were washed with PBS after 20 min and for two times. 2 N HCl was added and then removed after 20 min. Borate buffer was added and then washed with PBS. Afterward, 3% of Triton-X 100 was added to the plates and washed with PBS after 20 min. Next step was adding 10% normal goat serum and then removing it after 20 min. Then, the cells were incubated with polyclonal rabbit antibodies Bax (1:100, Santa Cruz Biotechnology, SC493) and polyclonal rabbit antibodies Bcl-2 (1:100, Santa Cruz Biotechnology, SC783) all over the night at 4°C. After the overnight incubation, coverslips were washed with PBS twice. The cells were incubated for 2 h with anti-mouse IgG (1:200, Abcam, Ab97022) and anti-rabbit IgG (1:200, Biorbyt, ORB180726) for 90 min in the darkness. Ultimately, the cells were washed with PBS, followed by the addition of PI for 20 min. The cells were visualized using a fluorescent microscope (Olympus Optical), and images were acquired with a magnification of ×400. Finally, the images were analyzed for markers' verification.

Caspase-3 and P53 proteins expression by flow cytometry

Flow cytometry measured the expressions of caspase-3 and P53 proteins. Cells (105) were poured into tubes, 4% paraformaldehyde was added to fix the cells, and the contents were washed with PBS after 20 min. Then, the cells were centrifuged (1200 RPM) for 5 min, followed by the addition and removal of PBS. 2N HCl was added for 20 min, followed by the addition of PBS and centrifugation for 5 min (1200 RPM). After that, 3% Triton X-100 and 10% goat serum were added. After 30 min, PBS was added, followed by centrifugation, and addition of monoclonal mouse antibodies: P53 (1:00, DAKO, CBL404), and polyclonal rabbit antibodies: Caspase-3 (1:00, Biolabs, 9662S) during the night at 4°C. The sample was centrifuged with PBS at 1200 RPM at 4°C for 5 min. Then, the cells were incubated with goat anti-mouse IgG (1:200 Abcam) and anti-rabbit IgG (1:200 Biorbyt) at 37°C for 90 min in the darkness. Afterward, the sample was transferred from the incubator to a dark room and was centrifuged with PBS at a temperature of about 4°C for 5 min (1200 RPM). Finally, the sample was examined by flow cytometry.

Statistical analysis

Data were expressed as mean ± SD, and standard error of the mean data normality was checked by the Kolmogorov–Smirnov test. In this study, the data were analyzed using a statistical software package SPSS Version 18.0 for Windows, International Business Machines COMPANY (IBM, Armonk, New York, USA). The differences were considered statistically significant, if there were P < 0.05 by an analysis of variance (ANOVA), followed by a Turkey's post hoc test. In this experiment, t-test and One-way ANOVA were used.


 > Results Top


Chlorogenic acid-inhibited growth of 4T1 breast cancer cell lines

As shown in [Figure 1], MTT assay, as an indicator for the survey of cell viability, proved that doses of 100, 150, and 200 μM of chlorogenic acid had a significant dose-dependent decrement in the cell viability of 4T1 cells, compared with the control cells. In addition, the inhibitory effect of chlorogenic acid has the high amount in 200 μM dose. The test was repeated three times, and identical results were obtained, to confirm the cytotoxic effects of chlorogenic acid.
Figure 1: Cell viability percentage analysis of 4T1 cells (by MTT assay). Comparison of cell viability percentage among the control groups with different doses of chlorogenic acid. Each value was reported as mean ± SD of three experiments, analyzed by analysis of variance followed by Tukey's post hoc test (*P < 0.05, ***P < 0.001)

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Chlorogenic acid-induced apoptosis in 4T1 cells

FITC-conjugated Annexin V-PI staining was used as a benchmark for identifying apoptotic cells, to determine the effects of chlorogenic acid on apoptosis induction and growth inhibition (find by flow cytometry). The control cells were negative for both Annexin V-FITC and PI. The analysis of other groups showed that in the early stages, apoptotic cell levels (Annexin V+/PI−) significantly increased from 1.14% to 0.04%, 34.56%, 40.9%, and 37.46% along with increasing concentration (0, 50, 100, 150, and 200 μM, respectively), while in the late stages, apoptotic cells ratio (Annexin V+/PI+) significantly increased from 2.34% to 2.08%, 13.45%, 17.7%, and 29.06% when the cells were treated with the concentrations of 0, 50, 100, 150, and 200 μM of chlorogenic acid, respectively [Figure 2]a and [Figure 2]b.
Figure 2: (a) Chlorogenic acid's ability to inducing apoptosis in 4T1 cell lines. Flow cytometry dot plot of 4T1 cells stained with Annexin V-FITC and propidium iodide. A. Untreated cells used as controls and B. 4T1 cells treated by concentrations of 50, 100, 150, and 200 μM of chlorogenic acid for 24 h (left to right, respectively). Treated cells had significantly increased rates of early and late apoptosis compared with the control. All dot plots were drawn by the Tree Star FlowJo X v10.0.7 R2-win software, Becton, Dickinson & Company (BD), Boston, USA. (b) Apoptotic cells percentage, based on the evaluation of apoptosis by Annexin-V-propidium iodide. Comparison between early and late apoptosis is a result of different concentration of chlorogenic acid on 4T1 cells' effect. Each value was reported as mean ± SD of the mean of three experiments. Data analyzed by analysis of variance followed by Tukey's post hoc test (*P < 0.05, **P < 0.01)

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Chlorogenic acid increased expression of Bax and decreased expression of Bcl-2 in the 4T1 cells

Immunocytochemistry results illustrated upregulation of Bax and downregulation of Bcl-2 in 4T1 cells that were treated with chlorogenic acid compared with the control group (76% ± 0.02 vs. 13% ± 0.02, P < 0.001 and 4% ± 0.03 vs. 70% ± 0.05, P < 0.001, respectively) [Figure 3]a and [Figure 3]b.
Figure 3: (a) Immunocytochemistry of Bax and Bcl-2 in 4T1 cells treated with chlorogenic acid. Bax and Bcl-2 staining were analyzed by immunocytochemistry using confocal laser scanning microscopy. The nuclei stained with propidium iodide (observed red). Arrows show the primary antibodies of Bax or Bcl-2 (observed green). The merged images show clearly protein expression of Bax or Bcl-2 (×400). (b) Comparison of proteins expression percentage of Bax and Bcl-2 in 4T1 cells treated with chlorogenic acid (200 μM) and untreated 4T1 cells. Treated cells have significantly increased the proteins expression of Bax and decreased the proteins expression of Bcl-2 compared with the control. Each value is reported as mean ± SD of three experiments. Data were analyzed by independent samples t-test (***P < 0.001)

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Chlorogenic acid increased the expression of P53 and caspase-3

Flow cytometry analysis showed that the 4T1 cells that were treated with chlorogenic acid significantly have higher levels of P53 and caspase-3 expressions compared with the control group (55.86% ± 7.12 vs. 7.37% ± 0.75, P < 0.001 and 55.86% ± 3.44 vs. 4.18% ± 3.70, P < 0.001, respectively) [Figure 4].
Figure 4: Effect of chlorogenic acid on proteins expression percentage of P53 and caspase-3 in 4T1 cells. (a) Flow cytometry of P53 and caspase-3 in 4T1 cells treated with chlorogenic acid (200 μM) compared to control. (b) Comparison between the proteins expression of P53 and caspase-3 in 4T1 cells treated with chlorogenic acid and untreated 4T1 cells. As shown, proteins' expression of P53 and caspase-3 significantly increased after treatment of 4T1 cells with chlorogenic acid, compared to control cells. Each value is reported as mean ± SD of three experiments. Data were analyzed by independent samples t -test (***P < 0.001)

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 > Discussion Top


For breast cancer patients, despite the availability of well-designed and effective therapeutic pathways, there is still the need for more drug research, exclusively regarding metastatic breast cancer patients, due to the high mortality rate and side effects of chemical drugs.

Polyphenols are natural compounds which have shown high antitumor activity in breast cancer, especially through a variety of mechanisms which are not yet fully understood. The inclusive modulation mechanisms of various oncogenic signaling cascades that effect on cell proliferation, apoptosis, and induction of cell-cycle arrest.[22],[23],[24] In the meantime, chlorogenic acid is a known polyphenol that is present in coffee abundantly.[25] This antitumor compound[26] induces apoptosis through various pathways in different tumor cells.[18],[27],[28],[29] On the other hand, there are several molecular mechanisms that tumor cells use to suppress apoptosis and progress the tumor.[30] The β-catenin is a multifunctional protein with a key role in physiological homeostasis. Its improper high expression leads to several diseases including cancer.[31] Xu et al. reported antitumor molecular mechanism of chlorogenic acid on GSK-3β and APC genes inducing and β-catenin gene inhibiting.[32] Furthermore, it is proven that PI3K/AKT/mTOR signaling pathway plays a major role in virulent conversion, and in consecutive growth, proliferation, and metastasis of human tumors.[33] Due to this, Refolo et al. have shown that chlorogenic acid enhanced the anticancer effect of regorafenib through the inhibition increasing of MAPK and PI3K/AKT/mTOR signaling in human hepatocellular carcinoma cells.[34] Reactive oxygen species (ROS) are important sources of cellular oxidative stress conditions, which could be detrimental for normal cells, and they can cause DNA damage, genomic inconstancy, reprogramming cancer cell metabolism, and ultimately facilitate cancer cell growth.[35] Related to this subject, Yan et al. demonstrated that chlorogenic acid sensitizes hepatocellular carcinoma cells to 5-fluorouracil treatment by the suppression, the overproduction of ROS, and activation of ERK.[20] Mitochondrial membrane potential (ΛΨm) is an important parameter of the mitochondrial function in the mitochondrial apoptosis pathway.[36] Yang et al. suggested that chlorogenic acid induces apoptosis by reducing the levels of ΔΨm and increasing the activation of caspase-3 pathways in human leukemia U937 cells.[18]

The p53, Bax, Bcl-2, and caspase pathway is one of the most important pathways for apoptosis in cancer's research.[37],[38],[39] To the best of our knowledge, this is the first report in which the cytotoxic effects of chlorogenic acid have been investigated in the 4T1 breast cancer cells. In this research, we demonstrated the effect of chlorogenic acid on apoptosis inducing in 4T1 cells through the p53, Bax, Bcl-2, and caspase-3 pathway. Our findings proved that chlorogenic acid induced cell death in a dose-dependent manner so that the IC50 values of chlorogenic acid for 4T1 cells were found to be 200 μM during the 24 h of treatment.

The P53 is considered as an upstream factor, and caspase-3 is known as one of the final proteases in apoptosis induction.[21] Furthermore, the P53 gene was usually expressed in a very low level and high expression of it that can promote cell apoptosis.[40] The p53 tumor suppressor gene is strongly involved in DNA repair, cell cycle regulation, and programmed cell death. Furthermore, several studies suggested that p53 death signals lead to caspase activation,[38] and more than 50% of human cancers have a mutated nonfunctional p53.[39] Our results demonstrated that high levels of p53 expression were detected in 4T1 cells that were treated with chlorogenic acid. In another study, Magalhães DB et al. have shown that the inhibitory effects of Melissa officinalis L. ethanol extract were provided via increased level of P53 in MCF-7, AGS, and NCI-H460 cells.[27] Changes in levels expressions of Bax and Bcl-2 have been identified as a major pathway in apoptosis induction of the tumor cells;[41] as a result, it has been demonstrated that Bax upregulation and Bcl-2 downregulation attenuated cancer cells' growth.[19],[42] Our analysis illustrated that chlorogenic acid facilitated upregulation of Bax proapoptotic and downregulation of Bcl-2 antiapoptotic proteins in 4T1 cells. By many apoptotic factors which ultimately induce cell apoptosis work on downstream effector of caspase-3, it is activated in both endogenous and exogenous apoptosis pathways.[26] Eventually, this study demonstrated that the 4T1 cells treated with chlorogenic acid showed a high level of caspase-3 expression. All of the results showed a pathway for apoptosis induction in 4T1 cells that were treated with chlorogenic acid. However, according to the results of Annexin V/PI assay, we detected a significant high apoptosis induction in 4T1 cancer cells treated with chlorogenic acid. Generally, our findings are compatible with previous studies in this field, and it seems that chlorogenic acid induces increment of P53 and Bax expression and suppresses Bcl-2 expression. This function probably releases cytochrome C and finally increases caspase-3 expression, and induces apoptosis via the intrinsic apoptotic pathway after that, and inhibits tumor cell growth. However, more studies are needed for confirming this hypothesis.


 > Conclusion Top


This study demonstrated that chlorogenic acid plays an important role in apoptosis induction on the 4T1 breast cancer cells and can be considered as a good therapeutic agent candidate for the breast cancer.

Acknowledgments

We would like to thank Prof. Zahir M. Hassan, Dr. M. Dolati, Dr. Sh. Ababzadeh, Dr. M. Eslami Farsani, Dr. T. Komeili-Movahhed and A. Zayadi for their assistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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