Introduction

Glycine (NH₂CH₂COOH), which is unique because it has no isomeric forms and is a structural unit for many proteins, has been the subject of numerous studies (1). Sulfur-containing amino acids play major roles in synthesis, structure, and function of proteins (2). Sulfur is a critical element and it is found in the structure of cysteine and methionine, and it is  necessary for the biosynthesis of sulfolipids, antioxidants, cofactors, secondary metabolites and amino acids (3,4). Glutathione [g-glutamyl-L-cysteinylglycine; (GSH)] containing glycine and cysteine amino acids is the strongest antioxidant in the body. It is found in almost all tissues (at concentrations of 1-10 mM) (4-6). GSH is a necessary endogenous tripeptide for the prevention of neoplasms, liver disorders, corneal disorders, eczema, heavy metal poisoning, and to prevent the negative effects of radiation therapy (7,8). Supplementing of component amino acids (cysteine, glycine and glutamate) increases GSH synthesis in tissue. Studies have reported that high GSH concentration is effective against cellular damage, tissue degeneration and disease progression (8,9). GSH is a protein that fights oxidative stress and regulates cell proliferation, apoptosis, signal transduction, gene expression, insulin resistance, immune function and fibrogenesis (4,10,11). It has been reported that negative changes in GSH homeostasis cause obesity, cancer, AIDS, diabetes mellitus and heart disease (8,12) and GSH regulates cell death. GSH level affects the expression and activity of caspases and signal molecules that are important in cell death (12). GSH levels decrease during apoptosis. Many studies suggest that GSH output through the cell during apoptosis is associated with multiple drug resistance proteins (MRP) (4,13-15).

Several members of the MRP family, ABCC1 (MRP1), ABCC2 (MRP2), ABCC4 (MRP4), ABCC5 (MRP5) and ABCC7 (CFTR) have been shown to mediate GSH transportation (14). MRPs can transport structurally and mechanically different drugs, including natural anticancer drugs, nucleoside analogues, antimetabolites and tyrosine kinase inhibitors (16). MRPs are included in the ABCC subfamily of the ABC transporter family (17,18). Expression of ABCC proteins is seen in most human tissues (18). P-glycoprotein (P-gp; ABCB1), the first discovered member of the ABC (ATP-binding cassette) transporter family, is a membrane protein that works as an efflux pump (19-21). Proteins specific to breast tissue; ABCB1 (MDR1-P-gp), ABCC11 (MRP8), ABCC12 (MRP9), ABCG2 (BCRP) and colon tissue-specific proteins; ABCB1 (MDR1-P-gp), ABCC3 (MRP3), ABCC10 (MRP7), ABCC11 (MRP8), ABCC13 (Pseudogene), ABCG2 (BCRP) belong to the ABC family (18,22).

The p53 protein is a tumor suppressor gene consisting of 393 amino acids weighing 53 kDa. The p53 gene makes a choice between life and death (apoptosis) (23-25). Generally, p53 is upregulated or mutated in MRPs of cancer cells (26,27). Intracellular BCL-2/BAX ratio is extremely important in determining whether the cell will go to apoptosis. If BAX is too much, the cell will go to apoptosis, or if BCL-2 is too much, apoptosis will be inhibited (28-30). Activation of effector caspases such as Caspase-3 causes downstream of PARP and similar substrates. Thus, it initiates apoptotic cell death (31). PARP down regulation suppresses cell proliferation and causes apoptosis via the p53 signal pathway (32). HSPs play a role as molecular chaperones that ensure the correct folding of the newly synthesized proteins or stress-dependent misfolded proteins and also prevent aggregation of the proteins (33-35). Expression of HSPs plays a role in regulating apoptosis, immune response to tumors, and multiple drug resistance. Increased HSPs levels make cells more resistant to apoptosis (36,37). Glucose regulated proteins (GRPs) are molecular chaperones in cancer tissues that are upregulated by cellular response to various stressful conditions such as glucose deprivation, oxidative stress and hypoxia (38-40). At the present time, the elimination of drug resistance (41,42) and increasing the ability to direct resistant cells to apoptosis (43,44) are the main targets for research and development of anti-cancer agents.

In this study, it was aimed to investigate the effects of sulfur containing glycine imine derivatives compounds on MRPs and apoptosis mechanism in MCF-7 (breast cancer) and DLD-1 (colon cancer) cell lines.

In our previous study, three different compounds were synthesized from glycine imine containing sulfur. Antioxidant and cytotoxic activity studies of the compounds were carried out using five different methods. The compounds were shown to exhibit antioxidant and cytotoxic activities in MCF-7 and DLD-1 cells (45). As a continuation of the study, the effects of sulfur-containing glycine imine derivatives on MRPs and apoptosis mechanisms in MCF-7 (breast cancer) and DLD-1 (colon cancer) cell lines.  In our study, we showed that sulfur glycine imine-derived compounds had the ability to direct drug-resistant cells to apoptosis and exhibited significant activity in MCF-7 and DLD-1 cells.

Methods

In MCF-7 and DLD-1 cell lines; mRNA levels of MRPs (ABCB1, ABCC3, ABCC10, ABCC11 and ABCG2), apoptosis mechanism proteins (BAX, BACL-2, P53, PARP, CASP3), heat shock proteins (HSPs) and endoplasmic reticulum (ER) chaperone proteins (GRPs) were determined by qRT-PCR method.

Cell Culture

In this study, in vitro studies were used for MCF-7 (ATCC® HTB22™) (breast adenocarcinoma) DLD-1 (colon cancer) (ATCC® CCL221™) human cell lines. MCF-7 and DLD-1 cells were cultured using 25 g/100 mL sodium bicarbonate, 10% fetal bovine serum (FBS) RPMI-1640 (Roswell Park Memorial Institute) mediums containing 1 % penicillin/streptomycin in 25 cm² or 75 cm² flask, 5% CO₂ and it was produced by incubation for 24 hoursat 37 °C. Stock solutions were made in dimethyl sulfoxide (DMSO) (Sigma, Steinheim, Germany) and were made with culture medium. Controls were cultured in RPMI 1,640 medium with a final concentration of 0.1% of DMSO but without compounds (46).

qRT-PCR Assay

Total RNA Isolation

The MCF-7 (breast cancer) and DLD-1 (colon cancer) cells were incubated at 37 ^oC for 24 hours, they were added to the 6 plates using a number of 2x10⁶ cells. Cells with compound dose determined as 50 µM according to MTT results were washed with 1X PBS at the end of 24 hours and 1X trypsin was removed with EDTA. In order to evaluate expression at the gene level, mRNA extraction was studied in the control and dose groups in accordance with the method recommended in the kit (Thermo Fisher; RNA isolation kit [K0731]). The concentration and purity of the isolated RNA were carried out with the aid of the nanodrop device. In the process of measuring the RNA samples with nanodrop, the RNA samples were diluted at appropriate concentrations (ng/µL) and they were measured at 260 and 280 nm (µg/mL) = [optical density 260x dilution rate x 40 (µg/mL)] (A260/A280≅2.0). These RNA samples were included in the study.

cDNA Synthesis

The complementary chains of isolated total RNAs were converted into cDNA by synthesizing them with “Thermo Fisher; cDNA synthesis kit (K1622)” using oligo d (T) primer and reverse transcriptase enzyme (RT). In the 1^st stage of cDNA synthesis; it was kept at 65 °C for 5 minutes. In the 2^nd stage; it was kept for 1 hour at 42 °C and 5 minutes at 70 °C to inhibit the enzyme after incubation. Synthesized cDNAs were kept at -20 °C for qPCR.

qReal Time PCR (qRT-PCR)

The sequences of genes were determined by NCBI database. In line with the accessory numbers listed in the Table 1, the primers required for real-time PCR were designed with the help of the NCBI primary blast program (https://www.ncbi.nlm.nih.gov/tools/primer-blast). (Thermo Fisher; K0221); Syber Green Master Mix (2x) 5 µL Forward primer (10 mM) 1 µL, Reverse primer (10 mM) 1 µL, cDNA 2 µL and Nuclease free water 1 µL, using Piko Real 96 (Thermo Scientific) device and by applying the amplification program, mRNA expression levels were examined. All samples were studied in duplicate. After real time PCR, the melt curve analysis was performed and the specificity of the primers was tested.

Results

Chemistry

We performed this study to understand MRPs and apoptosis mechanism of ethyl 2-((bis(propylthio)methylene)amino)acetate (1), ethyl 2((bis(ethylthio)methylene)amino)acetate (2), and ethyl 2-((bis(methylthio)methylene)amino) acetate (3) on the two different cancer cell lines (Figure 1).

Glycineimine Derivatives Compounds Regulate mRNA Expression of MRPs

In MCF-7 cells; ABCB1 gene expression was reduced by 0.6-fold, ABCC10 gene expression by 0.6-fold, ABCC11 gene expression by 0.5-fold, and ABCG2 gene expression by 0.3-fold with compound I compared with the untreated control. ABCB1 gene expression was reduced by 0.6-fold, ABCC10 gene expression by 0.6-fold and ABCC11 gene expression by 0.4-fold with compound II compared with the untreated control. ABCB1 gene expression was reduced by 0.7-fold, ABCC10 gene expression by 0.4-fold, ABCC11 gene expression by 0.5-fold, and ABCG2 gene expression by 0.2-fold with compound III compared with the untreated control.

In DLD-1 cells; ABCB1 gene expression was reduced by 0.4-fold, ABCC10 gene expression by 0.6-fold and ABCG2 gene expression by 0.8-fold with compound I compared with the untreated control. ABCB1 gene expression was reduced by 0.2-fold and ABCG2 gene expression by 0.7-fold with compound II compared with the untreated control. ABCB1 gene expression was reduced by 0.7-fold, ABCC10 gene expression by 0.4-fold and ABCG2 gene expression by 0.2-fold with compound III compared with the untreated control.

Compound I reduced the expression of all other MDR genes except ABCC3 compared to negative control in the MCF-7 cells. It was found that expression was decreased in other genes except ABCC3 and ABCC11 in DLD-1 cells . Compound II reduced expression in only ABCB1 and ABCC11 genes by half in MCF-7 cells. In DLD-1 cells, expression was decreased only in ABCB1 and ABCG2 genes. Compound III reduced expression in ABCB1, ABCC11 and ABCG2 genes by half in MCF-7 cells. Expression in ABCB1, ABCC10 and ABCG2 genes in the DLD-1 cells decreased significantly (Figure 2).

Glycineimine Derivatives Compounds Regulate mRNA Expression of HSPs and Apoptotic Genes

Compared to untreated control; gene expression of BAX was increased by 2-fold and p53 by 4.3-fold, and gene expression of BCL-2 was decreased by 0.2-fold, PARP by 0.5-fold, HSP60 by 0.5-fold, HSP70 by 0.8-fold, and HSP90 by 0.7-fold with compound I in MCF-7 cells. Gene expression of p53 was increased by 3.2-fold and gene expression of HSP27 was decreased by 0.5-fold, HSP40 by 0.9-fold, and HSP90 by 0.7-fold  withcompound I in DLD-1 cells.

Compared to untreated control; gene expression of BAX was increased by 3-fold, Caspase-3 by 3-fold, p53 by 14.4-fold, and gene expression of BCL-2 was decreased by 0.8-fold, PARP by 0.2-fold, HSP60 by 0.3-fold, and HSP70 by 0.7-fold with compound II in MCF-7 cells. Gene expression of p53 was increased by 3.8-fold and gene expression of HSP40 was decreased by 0.8-fold, and HSP90 by 0.4-fold with compound II in DLD-1 cells.

Compared to untreated control; gene expression of Caspase-3 was increased by 2.7-fold, and p53 by 9.5-fold, and gene expression of BCL-2 was decreased by 0.9-fold, PARP by 0.6-fold, HSP40 by 0.5-fold, HSP60 by 0.4-fold, HSP70 by 0.8-fold, and HSP90 by 0.5-fold with compound III in MCF-7 cells. Gene expression of p53 was increased by 1.8-fold,  and gene expression of PARP was decreased by 0.3-fold, HSP70 by 0.5-fold, and HSP90 by 0.3-fold with compound III in DLD-1 cells.

Compound I increased the expression of p53, BAX, CASP3 and GRPs in the MCF-7 cells. BCL-2 reduced the expression of HSPs other than PARP and HSP27. Expression of p53 was increased in DLD-1 cells and the expression of HSPs was decreased except HSP60. Compound II increased the expression of p53, BAX and CASP3 in the MCF-7 cells. It only reduced HSP60 and HSP70 expressions. Expression of p53 was increased in DLD-1 cells. HSP40, HSP70 and HSP90 expressions were decreased. Compound III increased the expression of p53 and CASP3 in the MCF-7 cells. BCL-2 reduced the expression of HSPs other than PARP and HSP27. Expression of p53 was increased in DLD-1 cells. PARP, HSP27 and HSP90 expressions were decreased. Compounds (I, II and III) did not have a significant effect on GPRs genes’ (GRP78 and GRP94) expression (Figure 3).

Discussion

In our previous study, it was demonstrated that sulfur-containing glycine imine derived compounds had antioxidant and cytotoxic activities in MCF-7 and DLD-1 cell lines (45).  It was stated that the compounds gave parallel results with other studies in both antioxidant and cytotoxic activities. As a continuation of the study, the effects of sulfur-containing glycine imine derivatives on MRPs and apoptosis mechanisms were determined in MCF-7 (breast cancer) and DLD-1 (colon cancer) cell lines. It was observed that biological activity studies were limited in the literature reviews of glycine imine derived compounds containing sulfur. Biological activity studies with these compounds were carried out for the first time by our group.

GSH, (2S) -2-amino-5 - [[(2R) -1- (carboxymethylamino) -1-oxo-3-sulfanylpropan-2-yl] amino] -5-oxopentanoic acid, and tripeptide are found in all tissues. GSH consists of L-cysteine, L-glutamic acid and glycine, forming L-gamma-glutamyl-Lcysteinylglycine amino acids (47). GSH in cancer cells is important in the regulation of mutagenic mechanisms, DNA synthesis, growth, and multidrug and radiation resistance (48). GSH showed inhibition of apoptosis in HeLa (49), in HSC-2 cancer cell lines (50), and in Calu-6 lung cancer cells (51). GSH levels decrease rapidly during apoptosis, and this is known as a biochemical feature of oxidant-induced programmed cell death (52). GSH inhibits the induction of apoptosis in human lung cancer cells via down-regulation of survivin (inhibitor of the apoptosis protein family). In drug-resistant cells, GSH protects healthy cells from oxidative stress, but prevents induction of apoptosis in tumor cells (53). In addition, GSH describes a new pathway of apoptosis dependent on PKC-delta activation and independent of p53, Bcl-2 and Bax levels (54). Fico et al. (13), 2008 reported that 2-deoxy-D-ribose (dRib) induced apoptosis by activating an oxidative stress by consuming GSH.  In our study, the effects of sulfur containing glycine imine derived compounds on apoptosis mechanism proteins (BAX, BACL-2, P53, PARP, CASP3), HSPs and ER GRPs in MCF-7 and DLD-1 cell lines. The compounds decreased expression in anti-apoptotic (HSPs, BCL-2 and PARP) genes, while increased expression in pro-apoptotic (BAX, P53, PARP) genes. Compound I increased the expression of p53, BAX, CASP3 and GRPs in MCF-7 cells. BCL-2 decreased the expression of HSPs other than PARP and HSP27. Expression of p53 was increased in DLD-1 cell and expression of HSPs was decreased. Compound II increased p53, BAX and CASP3 expressions in MCF-7 cells, as well as decreased HSP60 and HSP70 expressions. In DLD-1 cells, p53 expression was increased, moreover HSP40, HSP70 and HSP90 expressions were decreased. Compound III increased p53 and CASP3 expressions in MCF-7 cells. BCL-2 effectively reduced the expression of PARP and HSPs. In DLD-1 cells, p53 expression was increased, furthermore PARP, HSP27 and HSP90 expressions were decreased. Decreasing anti-apoptotic gene expressions and increasing pro-apoptotic gene expressions drive cancer cells to apoptosis. The data of the study determined that the compounds decreased HSPs, BCL-2 and PARP gene expressions and increased BAX, P53, CASP3, PARP gene expressions, leading MCF-7, and DLD-1 cancer cell lines to apoptosis.

In studies, Hammond et al. (14), 2007 showed evidence that GSH was released through MRP1 during both internally and externally induced apoptosis.  In our study, the effects of sulfur containing glycine imine derived compounds multidrug resistance proteins (ABCB1, ABCC3, ABCC10, ABCC11 and ABCG2) in MCF-7 and DLD-1 cell lines were investigated. The compounds decreased expression in multidrug resistance (ABCB1, ABCC10, ABCC11 and ABCG2) genes. Compound I effectively reduced the expression of MDR genes in the MCF-7 cell compared to the negative control. Compound I reduced the expression of ABCB1, ABCCC10 and ABCG2 genes in DLD-1 cells compared to the negative control. Compound II reduced expression of ABCB1 and ABCC11 genes by half in MCF-7 cells . Expression of ABCB1 and ABCG2 genes was decreased in DLD-1 cells . Compound III halved the expression in the ABCB1, ABCC11 and ABCG2 genes in the MCF-7 cells. Expression in the ABCB1, ABCC10 and ABCG2 genes significantly decreased in DLD-1 cells.  The expression of MRPs in the cell membrane prevents cancer cells from drifting into apoptosis. With the compounds used in the study, the expression of proteins that caused drug resistance was suppressed and it was proven that cancer cells could drift into apoptosis.

Conclusion

In brief, in our study, compounds increased gene expression in MDR and apoptosis mechanism genes (BAX, P53, PARP). HSPs and BCL-2 and PARP gene expressions decreased. There was no significant decrease in gene expression of GRPs. The compounds (I-II-III) were shown to have remarkable effects on ABCB1, ABCC10, ABCC11 and ABCG2 genes. Compounds I, II, and III were found to have no effect on the ABCC3 gene in MCF-7 and DLD-1 cells. No significant effect of the compounds (I, II and III) on GPRs genes (GRP78 and GRP94) was detected. Compounds (I-II-III) were found to have significant effects on apoptosis mechanism genes and heat shock proteins (HSPs). Compared to DLD-1 cells, the effects of the compounds on MCF-7 cells were more marvelous.

According to the results of the used compounds on MDR in cells, they had the ability to direct cancer cells to apoptosis. The compounds used in the study were useful for anticancer studies. Derivatives of the compounds were thought to contribute to further studies. Our results showed that sulfur glycine imine derivatives had a potent ability to direct resistant cells to apoptosis, although MCF-7 cells exhibited remarkable activity.

Acknowledgments: The authors thank the Amasya University Central Research Laboratory (AUMAULAB) for their kind understanding of using their facilities.

Ethics

Ethics Committee Approval: The article is a cell culture study.  It does not require an ethics committee.

Informed Consent: No patients were used in the study.  Informed consent is not required.

Peer-review: Externally peer reviewed.

Authorship Contributions

Surgical and Medical Practices: S.M., B.Y., M.G., T.Y., Concept: M.G., T.Y., Design: M.G., T.Y., Data Collection or Processing: S.M., B.Y., M.G., Analysis or Interpretation: S.M., B.Y., M.G., T.Y.,  Literature Search: S.M., B.Y., M.G.,  Writing: S.M., B.Y., M.G., T.Y.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: This work was supported by Amasya University (FMB-BAP 18-0333).