Analysis of human aldehyde dehydrogenases (ALDH) gene expression pattern in breast cancer tissue samples: rutin-copper complex inhibit the breast cancer cell proliferation

Higher aldehyde dehydrogenases (ALDH) activity is one of the important signatures of breast cancer appearance and has been associated with poor prognosis. ALDH1A3 has been over-expressed in breast cancer patients. This study aims to analyze gene expression patterns of 18 ALDH isozymes in breast cancer tissue samples. It is carried out using a chip-based microarray, next-generation DNA sequencing of ALDH2 gene following in silico study to identify the natural products which act as inhibitors for over-expressed ALDH isoforms. The synthesis of rutin-copper complex and cell viability assay is carried out on MDA-MB-468 cell line. ALDH1A3 and ALDH18A1 have shown the highest positive mean fold of variation; whereas, ALDH2 and ALDH1A2 have shown the highest negative mean fold variation. In silico studies revealed that rutin has the highest binding affinity with both ALDH1A3 and ALDH18A1 and supported with IC50 value of rutin against MDA-MB-468 cells (144.50 μg/ml). Chemically synthesized rutin-copper complex significantly lowered the IC50 value to 119.40 μg/ml. The next-generation sequencing study provides the novel single nucleotide polymorphism (SNP) from T to G in the ALDH2 gene. The present study signifies that, along with ALDH1A3, ALDH18A1 also acts as a marker for breast cancer. Apart from that, inhibitors of ALDH1A3 and ALDH18A1 were attained. Perhaps the single nucleotide polymorphism (SNP) obtained during the mutation analysis may be the probable cause of the highest downregulation of ALDH2 in breast cancer.


Background
Breast cancer (BC) is the second most common cancer that leads to the death of women globally [1]. In India and other Asian countries, the incidence of breast cancer continue to be on the rise and every year nearly half a lakh women lose their lives due to this deadly cancer [2]. The BC tumors, which were not expressing estrogen (ER), human epidermal growth factor receptor (HER2) and progesterone receptor (PR) denote as triple-negative breast cancer (TNBC) [3]. A variety of factors associated with breast cancer like age, family history, lifestyle, hormonal regulation, alcohol consumption, and smoking are chiefly responsible for this pathogenesis [4].
There are several biomarkers used for diagnosis of breast cancer [5]. Over-expressed genes in early stages of breast cancer, such as tumor protein 53 (TP53), phosphoinositide-3-kinase-catalytic-alpha polypeptide (PIK3CA), human epidermal growth factor receptor 2 (HER2) ERBB2, fibroblast growth factor receptor 1 (FGFR1), myelocytomatosis oncogene (MYC), and phosphatase and tensin homolog (PTEN) [6] given in (Table 1) are extensively studied. In humans, aldehyde dehydrogenases (ALDHs) are mainly involved in aldehyde detoxification and are also important in the synthesis of retinoic acid [7]. They also play a vital role in alcohol metabolism by removing aldehydes [8]. Among 18 different isoforms of ALDH, the activity of each isoform differs from the other according to the localization and tissue distribution.
Several studies have reported that ALDH is a specific cancer stem cell (CSC) marker in solid tumors like breast, colon, and lung. The isoforms ALDH1L1, ALDH1A1, ALDH1, ALDH1A3, ALDH2, ALDH1A2, ALDH3A1, and ALDH7A1 have been associated with different types of tumors. Among these genes, ALDH1A3 which is predominantly expressed in kidney, salivary glands, stomach, and thus over-expressed in breast cancer [9]. Diverse chemotherapeutic treatments also interfere with increased activity of ALDH. Certain biomarkers that lead to breast cancer can be used for proper analysis and further treatment [10]. Recent data suggest that there is a high demand for naturally extracted bioactive compounds for their anticancer properties worldwide [11]. Few findings also suggest that some phytochemicals can stimulate apoptosis in malignant cells in both in vivo and in vitro [12].
The rutin (quercetin glycoside) has tremendous clinical applications which include anti-inflammatory, anti-oxidant, and inhibition of platelet aggregation [13]. Rutin acts as an adjuvant agent that increases the cytotoxic efficiency of two important chemotherapeutic drugs viz., cyclophosphamide (CYC) and methotrexate (MTX) in MDA-MB-231 breast cancer cells [14]. In our present study, we investigated the differential expression patterns of all isoforms of ALDH genes in human breast cancer tissue samples and also synthesized the rutin-copper complex to understand the differences in the efficiency of anticancer potentials. In silico molecular modelling studies lead to new innovation in designing novel drugs as this study requires less time and can be performed with large quantities of ligands. Besides, it is easy to compare and is found effective in screening [15]. In this study, we have used 12 natural bioactive compounds ( Table 2) against ALDH1A3 and ALDH18A1 proteins through computational in silico docking. Next-generation sequencing (NGS) was carried out for ALDH2 gene using illumina platform.  Bioruptor (Diagenode). Data of the breast cancer patients without any major clinical issues were included and nine tissue samples were collected from Dharwad, India. The tissue samples were stored at -70°C in RNAlater solution for further experimental use.

Selection of patient sample and materials used
Isolation of RNA and labeling the target RNA easy Minikit (Cat-74104) from Qiagen was used to isolate total RNA from tissue samples. RNA integrity analysis, cDNA synthesis, labeling, and microarray methods were carried out sequentially according to Kulkarni et al. [16].

Next-generation DNA sequencing
The next-generation DNA sequencing was performed at genotypic technologies private laboratory Bangalore. For this study nine breast cancer tissue samples were collected and used for sequencing of chromosome 12 of gene ID ENSG00000111275 using illumina sequencing platform. 100 bp reads of illumina reads were sequenced using Illumina GAIIx Analyzer. The alignment software Burrows-Wheeler Aligner (BWA version 0.5.9 r16) was used to perform the gapped alignment of illumina sequences against the reference sequence. Alignment software bowtie (version 0.12.7) was used to perform ungapped alignment of illumina sequences against the reference sequence. Samtools (version samtools-0.1.7a) 2 was used for calling SNPs. The procedure flow chart is given in Fig. 1.

Synthesis of rutin-copper complex
The stoichiometric mixture of rutin and copper chloride dehydrate of 1mmol and 1.5 mmol were used respectively in methanol solvent and reflux for 5 h. The resulting pale yellow precipitate was dried and washed three times with a mixture of 1:3 (ethanol-water) and was dried. The resultant was used for further experimentation and analysis [17].

Cell culture and treatments
Breast cancer cells (MDA-MB-468) were cultured in Dulbecco's modified eagle medium (DMEM) which was supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1× anti-myocotic solution. In each well of 96 wells flat bottom plate 200 μl of the cell suspension containing approximately 10,000 cells were seeded and cultured in CO 2 incubator at 37°C in 95% humidity by supplying 5% CO 2 . The 100-600 μg/ml concentration of rutin and rutin-copper complex samples was used in the treatment of cells. The IC 50 value for both rutin and rutin-copper complex was calculated after 24 h of experimental incubation time using GraphPad prism version 5.1 [18].

Molecular docking studies
The molecular docking studies were performed on 12 natural compounds against ALDH1A3 (5FHZ) and ALDH18A1 (Protein data bank PDBID-2H5G) using Schrödinger Maestro 11.2 version. The 3D structure of the protein ALDH1A3 (PDB ID-5FHZ) was retrieved from PDB. The protein was optimized accordingly, hydrogen was added and water components surrounding greater than 5 Å were removed. Finally, it was minimized to the default root mean square deviation (RMSD) value of 0.30 using protein preparation wizard. Receptor grid generation was performed for minimized protein to identify the binding site of the ligand to the protein. The structures of natural compounds were drawn using 2D sketcher. They were performed using the Ligprep module to generate the 3D tautomer of the compound and to generate all combinations up to 32 per ligand. Ligand docking was performed according to the method of Kalirajan [19]. In Maestro 11.2 version dock, the binding of natural ligands to the active site of the human ALDH1A3 is an automated process with extra precision (XP) descriptor information and added epik state penalties to docking score. The best bioactive conformation was finalized based on the highest G score and highest number of interactions with the protein, mostly the number of hydrogen bonds. The XP visualizer was used as a tool to scrutinize the interactions. The scoring method was the most accepted design, with different parameters like polar, salvation, repulsive, hydrophobic, electrostatic, activity, and G score.

Results
The microarray analysis has given an insight on differential expressions of ALDH isoforms in breast cancer tissues. ALDH1A3 and ALDH18A1 with highest positive mean fold variation and ALDH2 with highest negative mean fold variation were noted as depicted in Table 3. These results suggest that ALDH1A3 and ALDH18A1 act as biomarkers for breast cancer. Thus, these may be targeted further for designing inhibitors. ALDH1A3 and ALDH18A1 with 2.22 and 1.93 fold increases respectively were observed. In contrast, ALDH2 with − 4.19 fold decrease in expression made us curious to study the DNA sequence of ALDH2.

Next-generation sequencing (NGS)
To date, hardly any studies examined the role of ALDH2*2 in the development of breast cancer and none of them found any association between ALDH*2 in correlation with the risk of breast cancer [20]. These studies have shown G to A point mutation in ALDH2*2. However, in our study, a different novel SNP 'T to G point mutation' was observed in breast cancer samples and is depicted in Table 4. Effect of rutin and rutin-copper complex on cell viability was analyzed through in vitro studies. The analysis was carried out on MDA-MB-468 cells using cell viability assay 3-(4,5-Dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT assay) for different time periods. The IC 50 value of rutin was 144.50 μg/ml and the rutincopper complex was 119.40 μg/ml in MDA-MB-468 cells, with a difference of 25.10 μg/ml (Fig. 2). The lesser IC 50 value of the rutin-copper complex may suggest a suitable replacement of the rutin to act as an adjuvant.
Molecular docking studies have revealed that rutin had a best binding affinity with both ALDH1A3 and ALDH18A1 among different natural bioactive compounds as shown in Figs. 3 and 4. Schrödinger maestro docking scores of ligands against ALDH1A3 and ALDH18A1 are given in (Tables 5 and 6), respectively.

Discussion
Aldehyde dehydrogenases have a variety of biological effects. It has specifically been linked to different types of cancer, Parkinson's disease, cataract, obesity, etc. ALDH1A1 is highly expressed in tissues but not true in case of breast cancer. ALDH1A3 is a marker for breast cancer patients [21]. In the present study, ALDH1A3 gene was highly expressed compared to normal as reported earlier [22] but ALDH18A1 also exhibited high expression in breast cancer. This ultimately proves that ALDH18A1 also acts as a biomarker along with ALDH1A3. Hence, identifying inhibitors against these may help in better management of breast cancer.
Currently, chemotherapy is widely used to treat a variety of cancers which has its limitations with high toxic side effects to the patients [23]. Hence, research on an alternative approach to treat cancer without side effects is in high demand. Few natural products like flavonoids and polyphenols have better anticancer activity [24]. In silico molecular docking study revealed that rutin has higher binding affinity to both over-expressed ALDH1A3 and ALDH18A1 gene among 12 selected natural compounds.
As previously reported, the synergistic action of rutin (glycoside) with other chemo drugs, manifests its potential role as an adjuvant for the treatment of breast cancer patients. Recent study reports on flavonoids complexing with macromolecules to enhance their bio-availability and chemo-preventive efficacy. Rutin (Ru)-fucoidan (Fu) complex was used to overcome the limitations of bio-availability of rutin molecule [25]. Hence, in our current study, a rutincopper complex was synthesized to study the effects of both rutin and its complex with copper for their potential bioactivity against breast cancer cells. We noticed that the IC 50 value decreased to a large extent for rutin-copper complex compared to rutin. Hence, it suggests that the rutin-copper complex is a lead molecule for further studies in replacing rutin to act as an adjuvant in treating breast cancer patients. Further in silico molecular dynamics simulation study and in vivo studies also act as supportive information to understand the nature of rutin-copper complex. A rare mutation lys/lys genotype of rare SNP rs671, which is associated with increased breast cancer in Asian women, has been recently reported [26]. However, in this study, the expression of ALDH2 was very low in breast cancer tissue samples compared to normal breast tissues. Further, it showed different novel SNP with T to G rather than G to A.

Conclusion
Like ALDH1A3, ALDH18A1 also acts as a biomarker for breast cancer and a novel ALDH2 single-nucleotide polymorphism may also be a prognostic indicator for breast cancer in south Indian women. The computational study reveals that Rutin has greater binding effect on both ALDH1A3 and ALDH18A1 proteins. The rutin-copper complex has a greater effect on breast cancer cell lines as compared to rutin alone. These results should be further validated with in vivo studies and this study needs further investigation with a large number of patient samples.