Memantine induces apoptosis and inhibits cell cycle progression in LNCaP prostate cancer cells

Abstract

Deregulated cancer cell metabolism plays an important role in cancer progression. Cancer cell metabolism has been in the centre of attention in therapeutical cancer cell targeting. Repurposed chemical agents, such as metformin and aspirin, have been studied extensively as preventive and therapeutic agents. Metformin is Food and Drug administration (FDA)-approved antidiabetic drug cheaper than other chemotherapeutic agents that were shown to have anticancer effects. Memantine is an FDA-approved Alzheimer’s drug. Drug repositioning studies offer wide range of benefits, such as reduced time, cost and risk over de novo drug discovery. Therefore, we aimed to target glucose and glutamine metabolism in androgen-dependent LNCaP cells by using metformin and memantine and investigate these agents’ effects on prostate cancer cell proliferation in vitro. We evaluated the effects of metformin and memantine on the protein expression levels of genes that play significant roles in apoptosis and cell cycle progression (Casp3, Casp9, Bcl-2, Survivin, Bax, c-Myc, HIF1A, CCND1, CDK4 and GAPDH) by Western blotting. Alzheimer’s drug memantine exerted cytotoxic effects at 0.25 mM and metformin at 2.5 mM. We identified for the first time that memantine exerts antineoplastic activity (0.25 mM) by triggering Bax-dependent pathway of apoptosis. In addition to that both molecules have shown similar patterns on pro- and anti-apoptotic protein expression levels, such as Bcl-2, Casp3, Survivin and Bax. Our preclinic results indicate that memantine might be used as a new repositioned drug in cancer treatment. Beyond targeting glucose metabolism, glutamine metabolism also holds great promise for a potential treatment option.

Introduction

All cancer cells require changes in metabolism to support their growth and survival. Therefore, targeting cancer cell metabolism holds great promise as a novel anticancer strategy. ¹ Oncogenic transformation requires increased cell proliferation that enhances more nutrient and energy. Mutations that enrolled in this metabolic process can interact and participate in the malignant transformation. ² Cancer-related driver mutations affect core signalling pathways and processes responsible for tumorigenesis. Many critical oncogenic signalling pathways swift their mechanisms to adapt their growth and survival rates. ³ Addiction to glucose and glutamine becomes part of the oncogenic process; therefore, targeting and interfering glucose and glutamine metabolism might be a selective way of killing cancer cells. ⁴ Although glucose has already been a focus of attention for many years in the study of cancer metabolism, glutamine research has also gained interest in cancer cell metabolism studies due to its diverse range of cellular functions, such as energy production, macromolecular synthesis, reactive oxygen species homeostasis and the mammalian target of rapamycin activation. ⁵ Glutamine is the most abundant amino acid in blood. In cancer cells, it becomes a carbon source to support tricarboxylic acid (TCA) anapleurosis and biosynthesis reactions that are required to maintain proliferative phenotype. ⁶ Moreover, in many cancer cells, glutamine is the primary mitochondrial substrate and is required to maintain mitochondrial membrane potential and integrity. ⁷ Glutamine also serves as a nitrogen source required for the biosynthesis of purine and pyrimidine nucleotides. ¹ Deregulated glucose metabolism, fatty acid synthesis and serine–glutamine metabolism also contribute to the enhanced cell proliferation, survival, invasion and metastasis. ⁸

Instead of de novo drug discovery, the process of finding new uses different from the original medical indication termed ‘drug repurposing’ enables shorter pathways to the clinic. In drug repurposing applications, some of the steps can be bypassed like in vitro–in vivo screening, chemical optimization and toxicological studies as they have already been performed. ⁹

Repurposed metformin has been widely used in several applications like the prevention of cancer, as antidiabetics and in aging research. Several epidemiologic researches have indicated that metformin usage is associated with decreased risks of cancer incidence and mortality rates. ¹⁰ Metformin also decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism. ¹¹ Metformin is known to inhibit complex I of mitochondria that are prominent and attractive targets in treatment. ^12,13 Memantine is a blocker of N-methyl-d-aspartate receptor used in Alzheimer’s disease, and it increases complex I and decreases complex IV mitochondrial activity. ¹⁴ Memantine is used in targeting the effects of glutamate excitotoxicity in Alzheimer’s disease and other dementias. ¹⁵ Therefore, we thought that memantine could be used for blocking the effects of excess glutamate that boost cancer cell growth. In our study, we used metformin as glucose metabolism-interfering agent, while we used memantine as glutamate metabolism-interfering agent on androgen-dependent LNCaP prostate cancer cell line.

In this study, we investigated the protein expression levels of apoptotic genes Casp3, Casp9, Bcl-2, Bax and Survivin and some of genes that play role in cell cycle progression and cancer-related cell proliferation like c-Myc, HIF1A, CCND1 and CDK4.

Materials and methods

Cell culture and chemicals

Androgen-dependent prostate cancer cell line LNCaP was kindly gifted from Dr. Levent Turkeri. LNCaP cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, Missouri, USA). Cells were grown in an incubator with 5% CO² at 37°C. Metformin and memantine were obtained from Sigma-Aldrich (St Louis, Missouri, USA), and they were dissolved in sterile distilled water.

Cell cytotoxicity assay

In total, 3 × 10³ LNCaP cells were seeded into a 96-well plate in 100 μL of medium and cultured for 24 h. Various concentrations of metformin and memantine were added to the culture medium, and cells were further cultured. [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT solution (5 mg/mL in phosphate buffered saline [PBS]) was added to each well and after that plates were incubated for 4 h at 37°C. Dimethyl sulfoxide (DMSO) was added to each well to solubilize formazan crystals, followed by incubation at 37°C for 30 min. Absorbance ratio was measured on SpectraMax M3 (Molecular Devices, Silicon Valley, California, USA) microplate reader at 570 nm.

Western blot analysis

LNCaP cells cultured in T-25 flasks (Thermo Fischer Scientific, Waltham, Massachusetts, USA) were lysed in 100 μL of Radioimmunoprecipitation assay buffer (RIPA) buffer including 1 mM Phenylmethylsulfonyl fluoride (PMSF) (Roche Diagnostics, Mannheim, Germany). In this study, memantine is proposed as a potent antineoplastic agent. In humans, the half-life of memantine is 60–80 h; therefore, we chose 48 and 72 h to evaluate its effects on protein expressions. Protein concentrations were determined using Bicinchoninic acid assay (BCA) protein assay (Pierce, Rockford, Illinois, USA). Protein lysates (20 μg) were heated for 5 min at 95°C in Lithium dodecyl sulfate (LDS) nonreducing sample buffer (Pierce, Rockford, Illinois, USA) and then loaded on 10% tris-glycine SDS-PAGE gels, after that transferred electrophoretically onto polyvinylidene difluoride (Pierce, Rockford, Illinois, USA) membranes at 250 mA for 2 h. Membranes were blocked with 5% nonfat dry milk in tris-buffered saline-Tween 20 (TBST, 20 mM tris, pH 7.6, 137 mM sodium chloride and 0.1% Tween 20) for 1 h at room temperature and incubated overnight at 4°C with the antibodies specific to Casp3, Casp9, Bcl-2, Bax, Survivin, c-Myc, HIF1A, CCND1, CDK4 and GAPDH (Thermo Pierce, Rockford, Illinois, USA). Blots were washed three times subsequently with TBST. Protein bands were detected with horseradish peroxidase-conjugated anti-rabbit secondary antibodies (CST, Danvers, Massachusetts, USA) and visualized by Luminata Forte Western HRP Substrate (Merck Millipore, Darmtadt, Germany). Chemiluminescent signals of immunoblots were documented using Gel Logic 2200 Pro (Carestream Health, Rochester, New York, USA). The band density of specific proteins was quantified using Image J (NIH, Bethesda, Maryland, USA). Western blottings were done in triplicate (n = 3).

Statistical analysis

Experiments were performed at least three times (n = 3). Statistical analysis was performed using SPSS 21.0 software. Different protein expression levels were compared with nonparametric test, Mann–Whitney U-test. p < 0.05 was considered statistically significant. ¹⁶ Metformin and memantine IC50 values were calculated using Prism 7 (GraphPad).

Results

In order to shed light on the antioneoplastic mechanism of memantine, we compared its protein expression patterns with metformin, whose anticancer activity has been studied extensively. We compared glucose metabolism-interfering agent metformin and glutamine metabolism-interfering memantine’s effects on LNCaP cells. We found that both metformin and memantine shared similar patterns on the expression of apoptosis-related genes Casp3, Casp9, Bcl-2, Bax and Survivin, cancer-related cell proliferation c-Myc and HIF1A and cell cycle-related protein expression CCND1 and CDK-4 (Figure 3). The details also can be found in the online supplementary material.

Metformin and memantines’ effect on cell cytotoxicity

In order to determine metabolism- and mitochondria-interfering effects of metformin and memantine on LNCaP cells, cells were treated with 2.5–15 mM range of metformin and 0.25–5 mM of memantine. IC50 concentration for metformin (2.5 mM) and memantine (0.25 mM) was determined by MTT assay. As shown in Figures 1 and 2, memantine inhibited LNCaP cell proliferation in a concentration-dependent manner at 48 h at 0.25 mM (IC50) concentration. Metformin led to growth inhibitions at 48 h at 2.5 mM (IC50). Metformin and memantine at concentrations 2.5 and 0.25 mM, respectively, for 48 h significantly reduced cell proliferation. Memantine at 0.25 mM concentration inhibited cancer cell proliferation rate at nearly 10 times lower concentration than metformin.

Figure 1.

Metformin was tested over the range of 2.5–15 mM on LNCaP cells for 48 h. Cell cytotoxicity was determined by MTT assay. IC50 value was calculated as 2.5 mM (p < 0.01)

Figure 2.

Memantine was tested over the range of 0.25–5 mM on LNCaP cells for 48 h. Cell cytotoxicity was determined by MTT assay. IC50 value was calculated as 0.25 mM (p < 0.01).

Effects of metformin and memantine on pro- and anti-apoptotic protein expression levels

LNCaP cells were incubated with metformin (2.5 mM) and memantine (0.25 mM) for 48 and 72 h. Cell lysates were analysed by Western blotting. Metformin and memantine decreased the anti-apoptotic Bcl-2 and Survivin protein expression levels at 48 h. We found that Bcl-2 and Survivin protein expression increased to their initial levels again at 72 h. In accordance with the findings, executioner Casp3 levels were increased at 48 h and decreased at 72 h. However, pro-apoptotic Bax expression level increased gradually in a time-dependent manner. The Casp9 levels were increased at 0.25 mM memantine treatment at both 48 and 72 h (Figure 3).

Figure 3.

Effect of 48–72 h treatment with metformin (2.5 mM) and memantine (0.25 mM) on the expression levels of: apoptotic genes Casp3, Casp9, Bcl-2, Bax, Survivin; glutamine metabolism-related c-Myc and HIF1A; cell cycle progression-related CCND1 and CDK4. GAPDH protein expression was used as loading control. Protein expression levels were determined by Western blotting. All bands were quantified using Image J (NIH, Bethesda, Maryland, USA). Metformin and memantine affected protein expression levels (Casp3, Casp9, Bcl-2, Bax, Survivin, c-Myc, CCND1 and CDK4) as statistically significant p < 0.01.

Effects of metformin and memantine on cell metabolism and cell cycle progression-related protein expression levels

Both metformin and memantine treatment inhibited c-Myc protein expression at 48 h. But c-Myc protein expression increased at 72 h again. In addition to that HIF1A protein expression levels were decreased slightly at 48 h and increased at 72 h. In the present study, we found that metformin and memantine induce cell cycle arrest at G0/G1 phase by reducing CCND1 protein levels in LNCaP prostate cancer cells. Metformin and memantine increased CDK-4 protein expression level (Figure 3).

When comparing with untreated LNCaP cells, metformin and memantine affected the protein expression levels of Casp3, Casp9, Bcl-2, Bax, Survivin, c-Myc, CCND1 and CDK4) as statistically significant p < 0.01 (Figure 3).

Discussion

A better understanding of altered metabolism is an urgent need to unveil tumorigenesis. In the present study, we aimed to target tumour cell cycle metabolism with two different mitochondria-metabolism interfering agents; metformin and memantin. We aimed to interfere cancer cell glucose metabolism with metformin and glutamine metabolism with memantine in order to compare their effect on protein expressions. We evaluated these drugs’ effect on several proteins that have prominent roles in cell cycle progression.

Both metformin and memantine induced apoptosis by increasing Casp3 and Casp9. Metformin and memantine decreased anti-apoptotic Bcl-2 and Survivin protein expression levels, whereas they increased pro-apoptotic indicator Bax protein expression. The effect of metformin and memantine was transient on the protein expressions of Casp3, Bcl-2 and Survivin, but the effect of metformin and memantines on Bax protein expression gradually increased in a time-dependent manner. These findings suggest possible antineoplastic mechanism of action on apoptosis for both metformin and memantine.

In cancers, glutamine metabolism is highly regulated by several factors, such as MYC, p53, Ras and HIF. In some cancers, Myc amplification is involved in glutamine addiction. ¹⁷ Metformin and memantine inhibited oncogene c-Myc protein expression at 48 h. c-Myc protein expression levels were increased again at 72 h due to the half-life of the drugs. Metformin and memantine decreased HIF1A protein expression slightly. The reason behind this might be the role of HIF in glutamine metabolism that enhances the growth of tumours especially in vivo. ¹⁸ Metformin and memantine inhibited cell cycle progression by decreasing CCND1 and increasing CDK4 protein expression levels.

Several studies indicate antidiabetic drug metformin’s cancer cell proliferation inhibiting effect. ¹⁹ Metformin is a repositioned drug and it could offer a potential therapeutical opportunity in cancer treatment. ²⁰ Metformin’s anticancer monotherapy or adjuvant therapy potential is also being investigated in many ongoing clinical trials. ²¹ In this setting, mitochondria are provisioned as a prominent target for cancer therapy as being a central metabolic organelle required for tumorigenesis and being at the crossroads of life and death. ^22,23 Liu et al. also identified that metformin targets tumour cell intrinsic mitochondrial metabolism. ²⁴ Metformin is shown to inhibit complex I of the mitochondrial respiratory chain and reduce ATP synthesis. ¹² However, beyond this potential usage as a therapeutical agent, metformin is criticized for its high dosage needs to exert its anticancer activity in most of the preclinical studies. Due to this fact, there is a confusion about metformin’s clinically achievable concentrations and efficacy. ²⁵ Beyond that, the possible mechanistic effect of metformin as anticancer agent has been investigated intensely. In this study, we propose memantine as a potent antineoplastic agent whose IC50 levels were nearly 10 times lower than metformin.

Whether targeting cancer metabolism can provide a better option in cancer treatment question is still a hot debate. ‘Could metabolism be cancer’s Achilles heel?’ question still needs answers. ²⁶ Thus, a deeper understanding of the metabolic differences between normal and cancer cells need to be well understood so that these differences might contribute better treatment options, whereas most of the research that investigate cancer cell metabolism focus on glucose metabolism, targeting glutamine metabolism also holds great promise in cancer treatment. ⁵ In our study, we targeted glutamine metabolism with a potential repurposed drug memantine. We concluded that memantine might be used as an antineoplastic agent.

Drug repositioning studies offer great promise and lower R&D costs compared to de novo drug discovery. Memantine exerts its cancer cell proliferation inhibiting activity at 10 times lower concentration than metformin. Therefore, memantine could be a better choice for obtaining clinically achievable concentrations. To the best of our knowledge, this is the first study proposing memantine’s mechanism of antineoplastic activity by inhibiting CCND1 protein levels and inducing Bax-dependent apoptosis in LNCaP prostate cancer cells. As memantine stands out as a promising repositioned drug candidate, further in vivo studies and then the clinical trials are required in order to understand the implications of its various activities in the context of chemoprevention and adjuvant therapy.

Supplemental Material

Supplemental Material, Supplementary_data_(1) - Memantine induces apoptosis and inhibits cell cycle progression in LNCaP prostate cancer cells

Supplemental Material, Supplementary_data_(1) for Memantine induces apoptosis and inhibits cell cycle progression in LNCaP prostate cancer cells by G Albayrak, E Konac, AU Dikmen, and CY Bilen in Human & Experimental Toxicology