Investigating the Effects of Morin and Aminoimidazole Carboxamide Ribonucleotide on the AMPK/mTOR/ULK1 Signaling Pathway in Breast Cancer Cell Line (MCF-7)

Abstract

Background:

Breast cancer, one of the most prevalent cancers, remains the leading cause of mortality worldwide. Dysregulation of autophagy and apoptosis regulators is closely associated with cancer progression. AMP-activated protein kinase (AMPK) plays a critical role in tumorigenesis by regulating these processes. This study aimed to investigate the anticancer effects of Morin and aminoimidazole carboxamide ribonucleotide (AICAR) on viability, apoptosis, and autophagy in MCF-7 cells.

Materials and Methods:

MCF-7 cells were treated with different concentrations of Morin and AICAR for 24, 48, and 72 hours. Cell viability was assessed using the MTT assay to determine the effective Morin dose. Protein levels of UNC-51-like kinase 1 (ULK1), AMPK, mammalian target of rapamycin (mTOR), and LC3B/LC3A were analyzed by Western blotting. Reactive oxygen species (ROS) production was measured, and mitochondrial membrane potential was evaluated using rhodamine 123 fluorescence.

Results:

The (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay revealed that Morin (*P < 0.01), AICAR (*P < 0.01), and their combination (##P < 0.001) significantly reduced MCF-7 cell viability. Morin treatment markedly increased p-AMPK and p-ULK1 expression (*P < 0.05–***P < 0.001, #P < 0.05–##P < 0.01), and upregulated LC3B/LC3A (*P < 0.01–**P < 0.001, #P < 0.05), while p-mTOR expression was significantly downregulated (*P < 0.05–**P < 0.01, #P < 0.05). ROS generation in mitochondria increased markedly (*P < 0.01–**P < 0.001, #P < 0.01). Furthermore, Morin significantly decreased mitochondrial membrane potential (**P < 0.001–***P < 0.0001, #P < 0.01).

Conclusion:

Morin, reduced MCF-7 cell survival and proliferation, enhanced apoptosis, increased ROS production, and diminished mitochondrial membrane potential, confirming its potential anticancer role.

INTRODUCTION

Breast cancer is one of the most common types of cancer among women. This disease occurs when the cells in the breast grow abnormally and multiply out of control.[1] The factors engaged in the occurrence of breast cancer include age, pregnancy, family history, ethnicity, gender, and breastfeeding, hormonal replacement therapy, physical activity, and nutrition.[2] When cells do not have access to an adequate amount of nutrition or need more energy, they lose their cytoplasmic components and organelles through autophagy.[3] The accumulation of dysfunctional mitochondria contributes to oxidative stress, resulting to DNA damage, aging, and cancer.[4] Recent studies have shown that the loss of mitophagy (removing damaged mitochondria through autophagy) regulators function is closely pertaining to the expansion and progress of cancer.[5,6,7]

Among the molecules that have a major role in starting autophagy in breast cancer cells, mTOR (mammalian target of rapamycin), ULK1 (UNC-51-like kinase 1), (AMP-activated protein kinase), and LC3 (microtubule-associated protein one light chain 3) proteins are important.[8] Studies have shown that AMPK is an enzyme that promotes ATP production in response to a lack of nutrition. It can suppress cancer progression by inhibiting mTOR translation and synthesis that results in autophagy and apoptosis.[9] In contrast, mTOR suppresses autophagy, when it is going to occurs under cellular stress conditions. ULK1 is also an enzyme that acts as an autophagy originator and is phosphorylated by AMPK to operate autophagy, and mTOR also participates in the inhibitory phosphorylation of ULK1.[10]

Considering that the molecular mechanisms perform a notable role in the process of apoptosis and autophagy, researchers are trying to find compounds that affect these molecular mechanisms and then find ways to treat diseases such as breast cancer. In this study, we have reported the molecules AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) and Morin. AICAR (an AMPK activator) is a substance that has been studied for its potential therapeutic effects and the first identified AMPK agonist, which is utilized for activating AMPK in many in vivo and in vitro research works.[11,12]

Morin (3, 5, 7, 2′, 4′-pentahydroxyflavone; a yellowish pigment) is a bioflavonoid in several fruits and herbs. They are considered herbal medicines with several biological activities such as antioxidant cryoprotection, antimutagenics, and anti-inflammation.[13] Many studies have shown the anticancer potential of morin by suppressing growth and stimulating apoptosis in diverse cancer cell lines, for example, ovarian, lung, and colon cancer.[14,15,16] The present research was conducted to study morin and AICAR and their effect on the AMPK/mTOR/ULK1 signaling pathway in the breast cancer cell line (MCF-7).

MATERIALS AND METHODS

MCF-7 cell culture

MFC-7 cells were procured from (BON Yakhte Technology Co; Tehran, Iran) and cultured in RPMI plus FBS 10%, 2 mM L-glutamine, 2 g/L bicarbonate, and routine penicillin/streptomycin (BON Yakhte Technology Co; Tehran, Iran) in an incubator with 95% humidified air, 5% carbon dioxide, and 37°C. When the aggregation of cells gained 90%, it was thought out the optimal culture for the present research [Figure 1].

Figure 1.

The morphology and growth of MCF-7 cells after 2 days (a) and 4 days (b), (100×). Scale bar = 100 µm

Treatment of cells

Different concentrations of Morin (Solvent: DMSO) and AICAR (Solvent: DMEM) (sigma; USA) at 12, 24, 48, and 72 hours were evaluated by MTT assay to characterize the effectual doze. The IC50 of Morin was 50 µg/ml for 48 hours in this research after determining the concentration and effective duration of morin by MTT test and based on (IC50), the following groups were formed.

1. Control group: MCF-7 cells only received culture medium.

2. Morin group: MCF-7 cells were exposed to an effective concentration of Morin based on the results (IC50).

3. AICAR group: MCF-7 cells were exposed to 100 μM (64) of AICAR.

4. Morin group + AICAR group: MCF-7 cells received AICAR and Morin at the same time.

MTT assay

The MTT test is a quantitative test used to determine the impact of Morin and AICAR on the survivance of the MCF-7 cells. Briefly, cells were cultured in 96-well plates and 1 ml of 10% FBS medium was added to each surface. After 48 hours, 50 μL of MTT (Sigma, USA) at a concentration of 20 mg/mL was added to each well and incubated for one hour at 37°C. When the medium was removed, 700 μL of DMSO (BON Yakhte Technology Co; Tehran, Iran) was added to the wells. In the end, the absorption of the final solution was calculated with a spectrophotometer at a wavelength of 570 nm. Finally, the effective and appropriate concentration of morin and AICAR was calculated by GraphPad prism version 8.3.0 software.

Western blot analysis

The expression rate of ULK1, AMPK, mTOR, and LC3A/LC3B factors were determined by WB. After lysing, cells were centrifuged (12,000 rpm, 10 min) and subjected to SDS-PAGE and moved to polyvinylidene difluoride membrane. Following incubation (5% non-fat milk diluted in Tris-buffered saline + 0.05% Tween-20) for 2 hours, the membrane was incubated with primary antibodies at 1:200 diluted in Tris-buffered saline with Tween-20 and 5% BSA 0.05% overnight (4°C). The membranes were exposed to a secondary antibody (an anti-rabbit IgG at 1: 5,00) for 1 hour. The membranes were then extended by the enhanced chemiluminescence (ECL) reagents (Sigma, USA).

Measurement of mitochondrial ROS formation

2′,7′-Dichlorodihydrofluorescein diacetate indicator was used to measure the amount of ROS. This indicator penetrates the mitochondria and is hydrolyzed by the mitochondrial enzyme succinate dehydrogenase to 2’,7’-Dichlorofluorescein (sigma; USA), which is not fluorescent. After formation, the latter substance reacts with ROS inside the mitochondria and creates the compound 2’,7’-dichlorofluorescein, which is highly fluorescent. Following centrifugation of the mitochondrial suspension, dichlorofluorescein diacetate was mixed to the resulting precipitate. Following gentle mixing, incubation of the suspension was conducted for 10 min in a water bath with a temperature of 37. Then, the amount of ROS formation was determined with a spectrofluorimeter (LS50B, USA) based on the unit of fluorescence intensity. The emission and excitation wavelengths were 520 nm and 500 nm, respectively.

Isolation of mitochondria

In this step, the cells were rinsed using cold PBS and exposed to 5 ml of isolation solution for 20 minutes on ice. The isolation solution consisted of 250 mM sucrose (Sigma; USA) comprised of 20 mM HEPES-KOH (with a pH of 7.5) (Sigma; USA), magnesium chloride (1.5 mM), potassium chloride (10 mM), 1 mM EDTA (1 mM. Sigma; USA), EGTA, (1 Mm. Sigma; USA). The cells were homogenized and then centrifuged at 750 rpm for 10 minutes and the supernatant was centrifuged (12000 rpm, 30 min, at 4℃). The obtained pellet was suspended in PBS (BON Yakhte Technology Co; Tehran, Iran), stored in a −80 freezer, and kept until use.

Mitochondrial membrane potential measurement

The reissue level of rhodamine 123 cationic fluorescent probe was considered a measurement of potential of mitochondrial membrane measurement. To this end, after centrifuging the mitochondrial suspension, the supernatant was disposed of. Then rhodamine 123 was mixed to the sediment. The obtained suspension was incubated for 10 min at 37 degrees. Intensity of fluorescence at excitation wavelength of 490 nm and emission wavelengths 535 nm was measured. The difference between the fluorescence intensity of the sample and the control indicates the extent of mitochondrial damage.

Sample size calculation and sampling method

The samples, purchased from BON Yakhte Research and Technology Center (Tehran, Iran) were cultured and passaged in appropriate and standard conditions. After determining the concentration and effective duration of morin by MTT test, four groups were formed. All tests are reiterated 4 times.

Statistical analysis

One-way ANOVA followed by a non-parametric test were utilized for analyzing data and the outcomes were presented as the mean ± SD and P value < 0.05. The analyzes were repeated at four times.

RESULTS

MTT assay

MTT assay showed that Morin could decrease the viability of MCF-7 cells (P < 0.01). AICAR could significantly reduce the viability of the MCF-7 cells (P < 0.01). Simultaneous administration of Morin with AICAR significantly decreased the duplication of the MCF-7 cells contrasted to cells treated with Morin (P < 0.001) [Figure 2].

Figure 2.

Morin effects on viability percentage of the MCF-7 cells. Values are expressed as Mean ± SD (n = 5). *P < 0.01, **P < 0.001 (comparison with control), ^#P < 0.05 (comparison with Morin)

Western blot

Morin and AICAR was remarkably enhanced the production level of p-AMPK and p-ULK1 factors (P < 0.05), (P < 0.01), (P < 0.001), (P < 0.05) and (P < 0.01), but significant decrease of p-mTOR expression (P < 0.05), (P < 0.01) and (P < 0.05) [Figure 3]. Also, the LCB/LC3A ratio was significantly increased (P < 0.01), (P < 0.001) and (P < 0.05) in Morin, AICAR and Morin + AICAR groups [Figure 4].

Figure 3.

Western blotting results of different groups (Mean ± SD; n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.05, ##P < 0.01. Protein expression was semi-quantitatively calculated using ImageJ software

Figure 4.

Autophagy evaluation using LCB/LC3A ratio in various groups (mean ± SD; n = 5). *P < 0.01, **P < 0.001, ^#P < 0.05. Protein expression was semi-quantitatively calculated using ImageJ software

ROS formation

The ROS generation in MCF-7 cells mitochondria was considerably increased in Morin, AICAR and Morin + AICAR groups contrasted to the control grou (P < 0.001). Concomitant treatment of Morin with AICAR significantly accelerated the ROS generation compared to the Morin-treated cells (P < 0.001) [Figure 5].

Figure 5.

Results of ROS levels in the studied groups (Mean ± SD; n = 5). * Show a comparison with control and # comparison with Morin groups; *P < 0.01, **P < 0.001, ^#P < 0.01

Mitochondrial membrane potential

Morin and AICAR have remarkably decreased the mitochondrial membrane potential of MCF-7 cells (P < 0.001). In Morin, contrasted to the Control group, AICAR and Morin + AICAR groups, the mitochondrial membrane potential was significantly diminished (P < 0.0001) and (P < 0.01) [Figure 6].

Figure 6.

The expression percentage of Mitochondrial Membrane Protein in the control group and its comparison with the studied groups. The Mean ± SD are represented (n = 5). **P < 0.001, ***P < 0.0001 (comparison with control), ^#P < 0.01 (comparison with Morin)

DISCUSSION

Previous studies affirmed that natural products could exert many anti-cancer properties. In the present study, Morin remarkably decreased the survival rate and the proliferation of MCF-7 cells and inhibited cancer cells proliferation. Furthermore, Morin upregulated p-AMPK and p-ULK1 expression, and downregulated that of p-mTOR in the MCF-7 Cells. The AMPK/ULK1/mTOR path controls autophagy in reaction to anticancer factors.[17] The study of Aryal et al.[18] demonstrated that Baicalein triggered autophagic cell death thorough the AMPK/ULK1 pathway activation and modulating the expression of mTOR in prostate and breast cancer cells. Also, Xiang et al.[19] concluded that Fangchinoline could activate the AMPK/mTOR/ULK1 pathway in colorectal cancer Cells. Moreover, Furthermore, Zhang et al.[20] demonstrated that botulinic acid stimulates autophagy by activating the AMPK/mTOR/ULK1 pathway, which leads to an increase in p-AMPK and p-ULK-1 and a decrease in p-mTOR.

In this study, AICAR was used to examine the role of AMPK in the induction Morin-induced MCF-7 cell death. AICAR is generally uses as an AMPK activator in the study of the pathogenesis of cancers.[21] AICAR plays an important role in regulating cellular metabolism, exhibiting a wide range of therapeutic activities against various metabolic processes.[22] The present study proves that AICAR diminished the growth of MCF-7 cells. Morin with and without AICAR leads to a decrease in the level of p-mTOR and increases the rate of apoptosis in these cells. These data suggest that Morin is likely to be active as an AICAR activator in MCF-7 Cells. The most important mechanism used in the treatment of anticancer agents is apoptosis. The enhance of p-AMPK was associated with stimulation of apoptosis and decreased viability of MCF-7 cells exposed to Morin. AICAR is a potent activator of AMPK, a protein kinase that increases fatty acid oxidation in different tissues and stimulates glycolysis. Activation of AMPK by AICAR can induce apoptotic events. In addition, AICAR can also stimulate ERK1/2. ERK1/2 stimulated by AICAR is a protein involved in cell proliferation, and its regulation plays a wide and effective role in anticancer approaches. Therefore, AICAR can be considered as an important tool in cancer therapy.[23,24,25,26] Recent studies have confirmed that putting to work AMPK by AICAR causes to apoptosis in cancer cells.[27] Theodoropoulou et al.[28] investigated the effects of AICAR on retinoblastoma growth in mice. They observed that AICAR resulted in a 48% inhibition of retinoblastoma cell tumor growth in mice. Their results showed that AICAR treatment inhibited retinoblastoma tumor growth in vivo via the AMPK/mTOR pathway (AMPK activation and mTOR inhibition) and by apoptogenic, antiproliferative, antiangiogenic, and tumor vascular density mechanisms. Therefore, AICAR could be a promising new non-chemotherapy drug that may be effective as an adjuvant in the treatment of carcinoma.

It is considerable that, the AMPK/mTOR pathway is related to apoptosis and autophagy.[29] The study of Lee et al.[30] showed that Morin inhibited metastatic possible of MCF-7 human breast cancer cells through Akt/GSK-3β/c-Fos signaling pathway. In another study, Morin induced cell death by cell cycle suppression in MDA-MB-231 triple-negative breast cancer cells.[31] Also, Lee et al. evaluated the effects of Morin on metastasis and cell viability in HER-2-overexpressing human breast cancer SK-BR-3 cells. Their results showed that Morin prevents endothelial growth factor (EGF)-induced metastatic potential and suppresses cell migration and MMP-9 activity by inhibiting the EGFR signaling pathway in SK-B-3 cells. They reported that Morin also activated autophagy after 24 hour of treatment and this was maintained at 48 h.[32] A study showed that morin induces apoptotic cell death and autophagy in PC3 cells by activating AMPK and ULK1 and suppressing mTOR pathways.[33] Taken together, the present research suggests that Morin could be a powerful therapeutic candidate for the treatment of breast cancer because it induces cell death and suppresses metastatic potential by suppressing mTOR and stimulating AMPK.

CONCLUSION

Morin as a bioflavonoid, effectively decreased the survival rate and MCF-7 breast cancer cells proliferation. It also induced apoptosis, enhanced production of ROS and lowered the potential of mitochondrial membrane of cancer cells.

Limitation

There was no limit.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.