Fetal Hemoglobin Inducer Activity of Moringa oleifera, Curcuma aueruginosa Roxb., and Artocarpus altilis Based on the Gamma Globin Expression

Nisa Widya Amanda; Ita Margaretha Nainggolan; Irmanida Batubara; Uus Saepuloh; Huda Shalahudin Darusman

doi:10.4103/joah.joah_74_22

ORIGINAL ARTICLE

Year : 2023 | Volume : 14 | Issue : 1 | Page : 17-21

Fetal Hemoglobin Inducer Activity of Moringa oleifera, Curcuma aueruginosa Roxb., and Artocarpus altilis Based on the Gamma Globin Expression

Nisa Widya Amanda¹, Ita Margaretha Nainggolan², Irmanida Batubara³, Uus Saepuloh⁴, Huda Shalahudin Darusman⁵
¹ Department of Biotechnology, Bogor Agricultural University, Bogor, Indonesia
² Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Bogor; Department of Clinical Pathology, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, Jakarta, Indonesia
³ Tropical Biopharmaca Research Center, LPPM of Bogor Agricultural University; Department of Chemistry, Faculty of Mathematics and Natural Science, Bogor Agricultural University, Bogor, Indonesia
⁴ Department of Anatomy, Physiology and Pharmacology, School of Veterinary Medicine and Biomedical, Bogor Agricultural University, Bogor, Indonesia
⁵ Department of Anatomy, Physiology and Pharmacology, School of Veterinary Medicine and Biomedical, Bogor Agricultural University; Primate Research Center, LPPM of Bogor Agricultural University, Bogor, Indonesia

Date of Submission	21-Aug-2022
Date of Decision	29-Oct-2022
Date of Acceptance	15-Nov-2022
Date of Web Publication	17-Feb-2023

Correspondence Address:
Dr. Ita Margaretha Nainggolan
School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, Jl. Pluit Raya 2, North Jakarta, DKI Jakarta 14440
Indonesia

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/joah.joah_74_22

Abstract

BACKGROUND: Beta (β)-thalassemia is a common single-gene blood disorder resulting in a decrease or absence of β-globin chain synthesis. In β-thalassemia major, an imbalance of α/β globin chains results in severe oxidative stress that leads to pathological conditions. Reactivating the gamma (γ)-globin gene will overcome the excess alpha (α)-globin chains and relieve β-thalassemia patients' clinical course.
AIMS AND OBJECTIVES: This study aimed to determine the most competent herbal extract with high efficiency in inducing γ-globin gene expression to facilitate β-thalassemia patients.
MATERIALS AND METHODS: This study used K562 cell line culture to assess the fetal hemoglobin (HbF) induction efficacy of Moringa oleifera (MO), Curcuma aueruginosa Roxb. (CA), and Artocarpus altilis (AA). We carried out the benzidine test to count hemoglobin-containing cells and RT-qPCR to measure γ-globin gene expression.
RESULTS: The benzidine test showed that MO extract was the highest value (3%) in inducing fetal hemoglobin. However, based on RT-PCR analysis, CA extract had the most significant (2.39 fold change) ability to reactivate the γ-globin gene compared to Hydroxyurea as a positive control.
CONCLUSION: The different properties and levels of MO and CA testing in antioxidant and reactivation of gene expression mechanism most probably influenced the discrepancy between the benzidine test and RT-qPCR results.

Keywords: Beta-thalassemia, gamma-globin, fetal hemoglobin, herbal extract, K562 cell line

How to cite this article:
Amanda NW, Nainggolan IM, Batubara I, Saepuloh U, Darusman HS. Fetal Hemoglobin Inducer Activity of Moringa oleifera, Curcuma aueruginosa Roxb., and Artocarpus altilis Based on the Gamma Globin Expression. J Appl Hematol 2023;14:17-21

How to cite this URL:
Amanda NW, Nainggolan IM, Batubara I, Saepuloh U, Darusman HS. Fetal Hemoglobin Inducer Activity of Moringa oleifera, Curcuma aueruginosa Roxb., and Artocarpus altilis Based on the Gamma Globin Expression. J Appl Hematol [serial online] 2023 [cited 2023 Apr 1];14:17-21. Available from: https://www.jahjournal.org/text.asp?2023/14/1/17/369844

Introduction

Beta (β)-thalassemia is the most common monogenic blood disorder and globally has a high mortality rate.^[1] This red blood cell disorder results from a lack or absence of synthesis of β-globin chains, which are the main components in the composition of adult hemoglobin.^[2] As a result, an imbalance between the alpha (α)/β globin chains results in excessive accumulation of α globin chains. Aggregates of unbound α-globin cause an increase in oxidative stress so that the erythropoiesis process in the bone marrow becomes ineffective and can also shorten the lifespan of red blood cells. The presence of oxidative stress causes an increase in excessive iron levels in the body and plays an essential role in the pathophysiology of thalassemia and its complications.^[3] Some effects can also lead to pathological changes such as growth retardation, osteoporosis, heart failure, and other complications.^[4]

Fetal hemoglobin (HbF) is the Hb synthesized in every human that usually occurs in the early period of life. Pharmacologically, reactivation of the gamma (γ)-globin gene can increase the γ-globin synthesis and subsequently amount of HbF to compensate the decrease of β-globin synthesis, which results in ineffective erythropoiesis. Therefore, therapy with induction of HbF is considered the most appropriate treatment for β-thalassemia.^[5] Clinically, hydroxyurea (HU) is the only drug approved by the Food and Drug Administration as an HbF inducer in β-hemoglobinopathies, but its ability decreases in treatment and can cause infertility and teratogenicity in patients.^[6],[7] In addition, other available treatments are scheduled blood transfusions and the administration of chelating agents to prevent iron overload and bone marrow transplantation.^[1]

In recent years, several drugs have been identified for synthesizing HbF, such as HU and 5-azacytidine. However, their myelotoxicity and carcinogenesis effects encourage researchers to find other new compounds with high efficiency and accuracy in increasing HbF. Natural ingredients that plants own have become a source of compounds that are very important in treating various diseases. Phytochemical studies on Moringa oleifera (MO), Curcuma aeruginosa (CA), and Artocarpus altilis (AA) based on multiple extractions, isolation, and analytical methods showed the identification of flavonoids. The research explained that flavonoid compounds could be HbF-inducing agents.^[8],[9],[10] The other finding elucidated that quercetin, one of the flavonoid families, can overcome β-thalassemia through antioxidant pathways.^[11]

Subjects and Methods

K562 cell culture

Human K562 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 20% fetal bovine serum, 1% L-glutamine, and 1% antimycotic-antibacterial. Using a cell counter, we determined cell growth according to the number of cells/ml. We incubated the K562 cells for 2-3 days. We carried out the cell passage when the cells had 70%–80% confluence in the well plate. We calculated the cells using 0.4% trypan blue and a hemacytometer, then regrown in RPMI-1640 complete medium in a T75 flask and stored at 37°C incubator with 5% CO₂ for 3 days.

Trypan blue test

The trypan blue test determined the effect of the extract on cell growth and proliferation. Cell viability was determined on day five after herbal treatment by mixing cell suspension and trypan blue with a ratio of 1:1 volume and incubation time of 2 min at room temperature. We counted both living (unstained) and nonliving cells (blue) using a hemocytometer under inverted microscope. We use the following formula to calculate the cell viability and cell density as follows:

Testing of extracts on K562 cells

We use cells after passaging twice. Each sample extract was stocked with a concentration of 1000 g/mL and diluted to 10 g/mL. Before testing, we calculated the number of cells and added a uniform number of cells in each well plate. The number of cells to calculate cell viability and induction of HbF was 2 × 10⁴ cells/ml. The extract concentration was 0.1, 0.5, and 1 g/mL. We added each well plate with medium, cells, and extract. This experiment included the negative control dimethyl sulfoxide (DMSO) and the positive control HU. We incubated the cells at 37 °C with 5% CO₂ for 5 days.

Benzidine test

We prepared the benzidine stock solution by adding 1 g of benzidine dihydrochloride (Sigma-Aldrich, St. Louis, USA) to 14.5 ml of glacial acetic acid and topping up with 485.3 ml of distilled water. We made the working solution by putting together 20 ul of 30% H₂O₂ and 1 ml of the benzidine stock solution. Equal volumes of the working solution and cells in the tube were then incubated at room temperature for 2–3 min. Cells containing Hb are indicated by the appearance of blue color and then counted using a hemocytometer. Calculation of the percentage of positive Hb using the following formula:

Real-Time polymerase chain reaction

We extracted total mRNA from 5 days of incubating cells. The extracted RNA was partially diluted to obtain a uniform concentration between samples for cDNA synthesis. Reverse transcription was performed using random hexamers with the SYBR Green I reverse transcriptase and total RNA as a template. Real-time polymerase chain reaction studied the semiquantitative expression of γ-globin in HU and extracted treatments using glyceraldehyde-3-phosphate dehydrogenase (GADPH) expression as an internal control. Each recorded Cycle threshold (Ct) value is normalized for data.

Statistic analysis

All statistics were carried out by the software Statictical Package for Social Science (SPSS) by IBM, Chicago, USA. The statistical significances were calculated using a one-way analysis of variance, and P < 0.05 were considered statistically significant. The data are represented as mean ± standard deviation (SD).

Results

Effects of herbal extracts on K562 cell growth and viability

The inclusive inhibitory effect on the proliferation of K562 cells is presented in [Table 1]. The table presents no significant changes in cell viability after administering several herbal extracts with varying concentrations. It is known that different cell lines may show different sensitivity to certain compounds and concentrations. Thus, we investigated the relationship between the extract concentration and its cytotoxic effect on K562 cells by viability assay. The results of this experiment showed that the extracts of AA, MO, and CA in K562 cells showed values above 90%. After treatment with the extract, the cell culture showed no significant cytotoxic activity in K562 cells.

Table 1: Effects of extract for viability cell using trypan blue on K562 cells

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Effects of herbal extracts on K562 cell differentiation

The effect of erythrocyte differentiation after being incubated for 5 days is shown in [Table 2]. A benzidine stain test evaluated the accumulation of Hb formation in the cells. Almost all extracts have shown some degree of erythroid differentiation effect on K562 cells at concentrations of 0.1, 0.5, and 1 mg/ml in a dose-dependent manner. HU, an inducing agent, was used as a positive control with a maximum yield of 14.4%. The results found that HU showed a maximal Hb differentiation phenotype in K562 cells. Herbal extracts of MO, CA, and AA with various concentrations did not experience significant changes in K562 cells. Effect of the dose-dependent extract in erythroid differentiation of K562 cells showed MO extract at a concentration of 0.5 mg/mL induced erythroid differentiation with an increase of 3% ± 3.9% in benzidine positive cells. Similarly, AA extract at a concentration of 0.1 mg/mL showed a rise of 2.7% ± 3.5%. In comparison, CA extract at a concentration of 0.1 mg/mL showed an increase of 2.4% ± 1.1% in the benzidine-positive cell population.

Table 2: Effects of extract for induction of fetal hemoglobin using benzidine on K562 erythroid differentiation

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Herbal extracts induce γ-globin transcription and fetal hemoglobin expression in the K562 cell line

The transcription of the γ-globin gene was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) after being incubated for 120 h using herbal extracts. The graph in [Figure 1] shows that the herbal extracts used induced erythroid differentiation and hemoglobinization of K562 cells. We observed the effect of these extracts on the expression of the γ-globin gene associated with HbF production. The results showed that CA extract with a concentration of 0.5 mg/mL increased γ-globin expression by 2.39 times. AA extracts with a concentration of 1 mg/mL rise to 0.79 times, and AAs with a concentration of 0.1 mg/mL became 0.64 times, whereas HU showed a 1.95-fold increase in γ-globin expression in K562 cells during erythroid differentiation. The untreated control group showed no significant induction of γ-globin expression and normalized to one. The resultant data obtained were normalized with the housekeeping GADPH.

Figure 1: Level of γ-globin mRNA effects of several treatments natural herbal using RT-qPCR on K562 cells. CM = Complete Medium; HU = Hydroxyurea; DMSO = Dimethyl sulfoxide, MO = Moringa oleifera, CA = Curcuma aeruginosa, AA = Artocarpus altilis Roxb., RT-qPCR = Reverse transcription-quantitative polymerase chain reaction

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Discussion

We calculated the percentage of viability cells by giving trypan blue to cells with several concentrations of extracts used. Statistical tests showed that each group differed significantly, with a P < 0.05. These results indicated that the active extracts of AA, MO, and CA did not inhibit K562 cell proliferation in a concentration-dependent manner. According to the data, HU has a lower cell viability value than the others, 95.5% ± 2.3%. In addition, cell viability was more than 80%, suggesting that all natural extracts were less toxic than the positive control.

HU has been known to inhibit ribonucleotide reductase that have a toxic effect on cells.^[12] Leukemia cells incubated using MO extract at 800 and 1000 ug/ml gave better results in cell viability compared to those with 600 ug or lower.^[13]This is because the MO extract can reduce the lipid peroxidation process.^[13] Following the data obtained, the MO group treatment incubated for 120 h had a cell viability value of about 98%. The CA extract would experience K562 cell death at 25 g/ml.^[14] The last, AA extract, shows a decrease in cell proliferation with an increase in concentration.^[15] Concentrations between 50 g/ml and 75 g/ml are the most effective dose in decreasing number of living cells. This experiment used 0.1–1 mg/ml concentration, which did not reduce the viability cell value.

The results demonstrated that K562 cells experienced erythroid differentiation with various percentages of increase in benzidine-positive cells following treatment with several herbal extracts. Statistical analysis showed that each group differed significantly, with a P < 0.05. Furthermore, research was carried out using RT-qPCR to determine how much mRNA was produced in the γ-globin gene with the housekeeping gene GADPH. The accumulation of HbF is always accompanied by an increase in mRNA, meaning that the herbal extract used has the potential to be an anti-thalassemia drug. The results showed that the CA extract with a concentration of 0.5 mg/ml had the highest level in inducing mRNA γ-globin expression, as much as 2.39 fold changes. As for the positive control, the HU was 1.95 fold changes. One of the natural agents that could induce HbF was curcuminoid found in the rhizome of Curcuma.^[16] Curcuminoid could induce a 1.2 ± 0.4 fold change.^[17]

The positive control HU with a smaller value than the curcumin compound had a similar result to this current research.^[18] Several pathways can induce the formation of HbF, namely, Janus kinase/signal transducer and activator of transcription (JAK/STAT), mitogen-activated protein kinase, and phosphoinositide 3-kinase.^[19] Curcumin, a compound contained in the CA extract, can activate the JAK/STAT pathway. This pathway is triggered due to the binding of the hormone erythropoietin (EPO) with erythropoietin receptor (EPOR), resulting in the transphosphorylation of JAK2. Then, proceed with tyrosine phosphorylation of JAK2 and EPO-R in the cytoplasm. The resulting residue produces STAT5 monomers and migrates toward the target gene to the nucleus.^[20] A study carried out by Kalpravidh et al. showed that curcumin compound could improve the pathophysiology of β-thalassemia/HbE patients.^[21] The next highest extract value was AA extract, with 0.79 times the ability to produce γ-globin. This little value is probably because the chemical compound AA, with the majority of its content, quercetin, does not activate one of the essential pathways in the induction of HbF. The quercetin compound further activates the NRF2/KEAP 1 pathway, which this pathway has the primary function of an antioxidant.^[22] Quercetin from a group of flavonoid compounds is not capable of being one of the treatment agents for β-thalassemia.^[11]

Acknowledgments

We thank PT Industri Jamu Borobudur, Semarang, Indonesia for providing the herbal extracts. Also thank to Ms. Hanna Natalia and Ms. Almira Sidharta for technical assistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Ali H, Iftikhar F, Shafi S, Siddiqui H, Khan IA, Choudhary MI, et al. Thiourea derivatives induce fetal hemoglobin production in-vitro: A new class of potential therapeutic agents for β-thalassemia. Eur J Pharmacol 2019;855:285-93.
2.	Galanello R, Origa R. Beta-thalassemia. Orphanet J Rare Dis 2010;5:11.
3.	Yanpanitch OU, Hatairaktham S, Charoensakdi R, Panichkul N, Fucharoen S, Srichairatanakool S, et al. Treatment of β-thalassemia/hemoglobin E with antioxidant cocktails results in decreased oxidative stress, increased hemoglobin concentration, and improvement of the hypercoagulable state. Oxid Med Cell Longev 2015;2015:537954.
4.	Chumpuchanaphai S, Svasti S, Vadolas J, Thu YY. Screening of medical plants for fetal hemoglobin inducer as alternative treatments in β-thalassemia. Biomolecules 2021;2564:44-54.
5.	Khan F, Ali H, Musharraf SG. Tenofovir disoproxil fumarate induces fetal hemoglobin production in K562 cells and β-YAC transgenic mice: A therapeutic approach for γ-globin induction. Exp Cell Res 2020;394:112168.
6.	DeBaun MR. Hydroxyurea therapy contributes to infertility in adult men with sickle cell disease: A review. Expert Rev Hematol 2014;7:767-73.
7.	Flagnan JM, Howrad TA, Mortier N, Avlasevich SL, Smeltzer MP, Wu S, et al. Effect of rapamycin on accumulation of α-, β- and γ-globin mRNAs in erythroid precursor cells from β-thalassemia patiens. Mutat Res 2010;608:38-42.
8.	Farkhondeh T, Folgado SL, Pourbagher-Shahri AM, Ashrafizadeh M, Samarghandian S. The therapeutic effect of resveratrol: Focusing on the Nrf2 signaling pathway. Biomed Pharmacother 2020;127:110234.
9.	Perdhana IS, Suzana D. The role of quercetin on nrf2 expression in oxidative stress due to chronic kidney disease. Medical Informatics: Scientific Journal. 2019;2:27-36.
10.	Pace BS, Liu L, Li B, Makala LH. Cell signaling pathways involved in drug-mediated fetal hemoglobin induction: Strategies to treat sickle cell disease. Exp Biol Med (Maywood) 2015;240:1050-64.
11.	Sajadi Hezaveh Z, Azarkeivan A, Janani L, Hosseini S, Shidfar F. The effect of quercetin on iron overload and inflammation in β-thalassemia major patients: A double-blind randomized clinical trial. Complement Ther Med 2019;46:24-8.
12.	Theodorou A, Phylactides M, Forti L, Cramarossa MR, Spyrou P, Gambari R, et al. The investigation of resveratrol and analogs as potential inducers of fetal hemoglobin. Blood Cells Mol Dis 2016;58:6-12.
13.	Dilworth LL, Stennett D, Omoruyi FO. Effects of Moringa oleifera Leaf extract on human promyelocytic leukemia cells subjected to oxidative stress. J Med Food 2020;23:728-34.
14.	Fathonah F and Hardiani F. Activity of ethanol extract of temu ireng (Curcuma aeruginosa Roxb.) as antiproliferative in MCM/IPB-B3 and K562 tumor cells. Thesis 2015. Bogor: Faculty of Veterinary Medicine, Bogor Agricultural University; 2015.
15.	Jamil MM, Ganeson S, Mammam HB, Wahab RA. Artocarpus altilis extract on cervical cancel cells. Proceedings 2018;5:15559-66.
16.	Kukureja A, Wadha N, Tiwari A. Therapeutic role of natural agents in beta-thalassemia: A review. J Pharm Res 2013;6:954-9.
17.	Chaneiam N, Changtam C, Mungkongdee T, Suthatvoravut U, Winichagoon P, Vadolas J, et al. A reduced curcuminoid analog as a novel inducer of fetal hemoglobin. Ann Hematol 2013;92:379-86.
18.	Setyono F, Sadewa AH, Meiyanto E, Mustofa M. Curcumin and 6-Shogaol increase hemoglobin F levels by inhibiting expression of STAT3 mRNA gene in K562 line cell. Indian J Public Health Res Dev 2020;11:1191-6.
19.	Rahim F, Allahmoradi H, Salari F, Shahjahani M, Fard AD, Hosseini SA, et al. Evaluation of signaling pathways involved in γ-globin gene induction using fetal hemoglobin inducer drugs. Int J Hematol Oncol Stem Cell Res 2013;7:41-6.
20.	Swameye I, Muller TG, Timmer J, Sandra O, Klingmuller U. Identification of nucleocytoplasmic cycling as a remote sensor in cellular signaling by databased modeling. Proc Natl Acad Sci U S A 2003;100:1028-33.
21.	Kalpravidh RW, Siritanaratkul N, Insain P, Charoensakdi R, Panichkul N, Hatairaktham S, et al. Improvement in oxidative stress and antioxidant parameters in beta-thalassemia/Hb E patients treated with curcuminoids. Clin Biochem 2010;43:424-9.
22.	Gupta D. NRF2 Activation by KEAP 1 Inhibition Induces Fetal Hemoglobin and Regulates Antioxidant Response in Erythroid Cells. Dissertation. University of Miami; 2021.