The relative isoform expression levels of isocitrate dehydrogenase in breast cancer: IDH2 is a potential target in MDA-MB-231 cells

Shuyu Piao; Seonhee Kim; Youngduk Seo; Jinsun Lee; Sunhee Jeon; Giang-Huong Vu; Min-Kyung Yeo; Cuk-Seong Kim

doi:10.14216/kjco.23011

Korean Journal of Clinical Oncology > Article

Piao, Kim, Seo, Lee, Jeon, Vu, Yeo, and Kim: The relative isoform expression levels of isocitrate dehydrogenase in breast cancer: IDH2 is a potential target in MDA-MB-231 cells

Original Article

Korean Journal of Clinical Oncology 2023; 19(2): 60-68.

Published online: December 31, 2023

DOI: https://doi.org/10.14216/kjco.23011

The relative isoform expression levels of isocitrate dehydrogenase in breast cancer: IDH2 is a potential target in MDA-MB-231 cells

Shuyu Piao^1,^*

, Seonhee Kim^1,^*

, Youngduk Seo^2,^*

, Jinsun Lee^3,^*

, Sunhee Jeon¹

, Giang-Huong Vu¹

, Min-Kyung Yeo⁴

, Cuk-Seong Kim¹

¹Department of Physiology and Medical Science, Chungnam National University College of Medicine, Daejeon, Korea

²Department of Nuclear Medicine, Chungnam National University Sejong Hospital, Sejong, Korea

³Department of Surgery and Research Institute for Medical Sciences, Chungnam National University College of Medicine, Daejeon, Korea

⁴Department of Pathology, Chungnam National University College of Medicine, Daejeon, Korea

Correspondence to: Cuk-Seong Kim, Department of Physiology, Chungnam National University College of Medicine, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Korea, Tel: +82-42-580-8219, Fax: +82-42-585-8440, E-mail: cskim@cnu.ac.kr

^*Shuyu Piao, Seonhee Kim, Youngduk Seo, and Jinsun Lee contributed equally to this work as first authors.

Received July 14, 2023 Revised November 28, 2023 Accepted December 05, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Purpose

The isocitrate dehydrogenase (IDH) family plays an essential role in metabolism and energy production. The relative expression levels of IDH isoforms (IDH1, IDH2, and IDH3) have prognostic significance in several malignancies, including breast carcinoma. However, the IDH isozyme expression levels in different cancer stages and types have not been determined in breast carcinoma tissues.

Methods

We analyzed the messenger RNA (mRNA) and protein levels of IDH (IDH1, IDH2, and IDH3A) and α-ketoglutarate (α-KG) in 59 breast carcinoma tissues.

Results

The mRNA level of IDH2 was significantly increased at stages 2 and 3 in triple-negative and (ER-/PR-/HER+) breast cancers. However, the elevated α-KG level was only observed in stages 2 and 3, with no differences in the various breast carcinoma types. Western blotting analysis showed that IDH2 protein expression increased in the patient tissues and cell lines. An in vitro study showed IDH2 downregulation in the triple-negative breast cancer cell line MDA-MB-231 that inhibited cell proliferation and migration and induced cell cycle arrest in the G0/G1 phase.

Conclusion

These findings suggest that different from IDH1 and IDH3, IDH2 is more highly expressed in stages 2 and 3 breast cancer tissues, especially in triple-negative breast cancer. IDH2 potentially serves as a target to detect unknown mechanisms in breast cancer.

Keywords: Breast neoplasms, Isocitrate dehydrogenase, Isocitrate dehydrogenases 2, Cell proliferation

INTRODUCTION

Breast cancer is a common malignancy affecting women worldwide and is a major cause of cancer-related death in most countries [1,2]. Despite treatment with early diagnosis and the widespread use of improved adjuvant chemotherapeutic and hormonal agents, acquired resistance to therapy and disease recurrence remain a challenge [3]. Mitochondrial metabolism is essential for tumor proliferation, survival, and metastasis. The production of cellular energy and biosynthetic precursors are upregulated by the tricarboxylic acid (TCA) cycle, and these could be critical targets for cancer treatment. Like other cancers, breast cancer requires metabolic reprogramming to sustain its biosynthetic and bioenergetic needs, and these changes may lead to progression or recurrence. Therefore, we should focus on mitochondria as a therapeutic target in breast cancer treatment.

Isocitrate dehydrogenase (IDH) is a member of the IDH family. It is an important enzyme in mitochondria for energy metabolism in the TCA cycle and oxidatively decarboxylates isocitrate to α-ketoglutarate (α-KG). IDH consists of three isozymes: IDH1, IDH2, and IDH3 [4,5]. IDH1 is localized in the cytoplasm and peroxisomes, and IDH2 and IDH3 are localized in the mitochondria. IDH3 is a heterodimeric NAD⁺-dependent isoform and generates α-KG in the irreversible TCA cycle. However, mutations in IDH1/2 have been detected in various cancers and reduced α-KG to oncometabolite 2-hydroxyglutarate (2HG), which acts as an antagonist of α-KG to impair enzyme function in the TCA cycle. Although IDH1/2 mutations are thought to increase several malignancies, including breast carcinoma, overexpression of wild-type IDH1/2 was documented in esophageal squamous cell cancer, lung cancer, ovarian cancer, endometrial cancer, and advanced colorectal cancer [6–10]. Among these, overexpression of wild-type IDH2 in esophageal squamous cell cancer and lung cancer was significantly related to worse overall and progression-free survival [6,7]. This study focused on the relevance of the three wild-type IDH isoforms in breast cancers. Liu et al. [11] reported lower IDH1 expression in breast cancer patients was associated with higher snail protein expression. These characteristics could serve as a prognostic biomarker in breast cancer. Aljohani et al. [12] observed high expression of IDH2 immunoreactivity, an independent poor prognostic factor in breast cancer. In addition, Minemura et al. [13] compared the immunohistochemistry of three IDH isoforms in invasive breast cancer tissues and demonstrated that IDH2 was a potent prognostic factor in estrogen-positive breast cancer. Nevertheless, the expression of IDH isoforms in different cancer stages and types has not been thoroughly investigated in breast carcinoma tissues. Therefore, we compared the expression levels of wild-type IDH isoforms in 59 breast cancer patients’ tissues according to the stage and types. High IDH2 expression in stage III may be implicated in the development of breast cancer, and downregulation of IDH2 in MDA-MB-231 cells showed an anti-tumor effect in vitro.

METHODS

Cell culture and cell growth

Human breast epithelial cells (MCF10A), triple positive breast cancer cells (BT474), ER+/PR+/HER2− breast cancer cells (MCF7), ER-/PR-/HER2+ breast cancer cells (SKBR3) and triple-negative breast cancer cells (MDA-MB-231) were obtained from American Type Culture Collection (ATCC). MCF10A and BT474 were seeded onto 100 mm Falcon plates at 1×10⁵ cells/mL in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Gibco; Thermo Fisher Scientific, Inc.). MCF7 were seeded onto 100 mm Falcon plates at 2×10⁵ cells/mL in Minimum Essential Medium (MEM) supplemented with 10% FBS, 1% antibiotic-antimycotic, 1% MEM non-essential amino acid solution (Welgene, Cat.LS-005–01), 1% sodium pyruvate (Welgene, Cat.LS-013–01), and 10 μg/mL insulin. MDA-MB-231 were seeded onto 100 mm Falcon plates at 1×10⁵ cells/mL in RPMI 1640 supplemented with 10% FBS and 1% antibiotic-antimycotic. MDA-MB-231 were transfected with small interfering RNA (siRNA) targeting IDH2 (human siRNA sequence: sense, 5′-CAGUAUGCCAUCCAGAAGA-3′ and antisense, 5′-UCUUCUGGAUGGCAUACUG-3′) and negative control siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. These transfected cells were incubated for 2 or 7 days at 37 °C in a 5% CO₂ incubator. Cell counting was performed using ADAM MC Automatic Cell Counter (Bulldog Bio) according to the manufacturer’s recommendations.

Immunoblotting

MDA-MB-231 cells and cancer tissues were harvested and lysed in 100 μL RIPA buffer (1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.05 M Tris-HCl pH 8), containing 1× Halt protease inhibitor cocktail (Thermo Fisher Scientific) for 30 minutes on ice. After clearing by centrifugation at 13,000 rpm for 10 minutes, protein concentration of cell lysate was measured using a bicinchoninic acid (BCA) protein assay kit (iNtRON, cat. 21071). Equal amounts of protein per well were resolved via 10% to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was then washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) containing 0.1% Tween 20 (TBST) and blocked in TBST containing 5% bovine serum albumin Fraction V (Roche). Membranes were then incubated with the following antibodies: anti-p16 (sc-377412) and anti-p21 (sc-6246) (Santa Cruz Biotechnology); anti-IDH2 (ab129180), anti-IDH1 (ab230949), anti-IDH3A (ab228596) and anti-PINK1 (ab216144) (Abcam); anti-LC3 (NB100-2220, Novus Bio); anti-p62 (5114S) and anti-β-actin (4967S) (Cell signaling Technology). Immunoblotting of 30 μg of whole-cell lysate or tissue homogenate was performed similarly using appropriate primary and secondary antibodies. The membranes were treated with an appropriate peroxidase-conjugated secondary antibody, and the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate (Pierce Biotechnology). Values were normalized to β-actin as loading controls.

Real-time qPCR

Total RNA was isolated from cells or cancer tissues using the TRIzol method. Complementary DNA was generated from total RNA using the MAXIME RT Premix Kit (iNtRON Biotechnology). Relative RNA levels were detected by quantitative polymerase chain reaction (qPCR) using the Prism 7000 Sequence Detection System (Applied Biosystems) with the Super Script III Platinum SYBR Green One-Step qPCR Kit (Invitrogen). The primers were as follows: IDH1 forward 5′-GGC TTG TGA GTG GAT GGG TAA-3′ and reverse 5′-ACC TTT TGG GTT CCG TCA CTT-3′; IDH2 forward 5′-GAA GGT GTG CGT GGA GAC-3′ and reverse 5′-CCG TGG TGT TCA GGA AGT-3′; IDH3A forward 5′-GCC CAT TCC ACG ACG ACC AA-3′ and reverse 5′-TAG CAC CGC CAC TGC CAT CC-3′; and β-actin forward 5′-GGC TGT TGT CAT ACT TCT CAT GG-3′ and reverse 5′-GGA GCG AGA TCC CTC CAA AAT-3′. β-Actin was used as an internal control. Dissociation curves were generated to assess the aberrant formation of primer dimers. Relative expression levels were measured using the delta-delta Ct method.

α-KG assay

Breast cancer tissues were extracted and homogenized in assay buffer. The α-KG concentration was determined using an α-Ketoglutarate Colorimetric/Fluorometric Assay Kit (Cat. #K677-100; Biovision) according to the manufacturer’s instructions.

CCK-8 cell proliferation assay

Cells were transfected with small interfering IDH2 RNA (siIDH2) or small interfering control RNA (siCON) for 48 hours. Cell proliferation was then measured using a CCK-8 kit (Dojindo) according to the manufacturer’s instructions. Briefly, cells were washed with phosphate buffered saline (PBS) and resuspended in growth media including CCK-8 reagent added at 1/50 the media volume. These cells were then incubated at 37 °C for 1 hour in the dark. Cell proliferation was measured using an absorbance detector, with measurements performed at 450 nm.

Flow cytometry

Cells were transfected with siIDH2 or siCON for 48 hours. Cells were analyzed by flow cytometry for cell cycle using a NovoCyte flow cytometer, as indicated by the manufacturer (ACEA Biosciences). Cells were washed twice with PBS. These cells were fixed in 100% cold Ethanol for overnight at 4 °C, and stained with PI (5 μg/mL) and RNase A (1 mg/mL) in 500 °L PBS for 30 minutes at 37 °C. Flow cytometry data were analyzed using NovoExpress software.

Cell scratch assay

A wound healing assay was used to assess cell migration. Cells were transfected as described previously with siCON or siIDH2 in 6-well tissue culture plates for 24 hours, after which a sterile 200 μL pipette tip was used to detach the cells from the monolayer across the center of the well. Floating cells were flushed out by gently rinsing twice with PBS and replaced with serum-free medium (to rule out cell proliferation as the cause of wound closure) followed by incubation for another 24 hours. The total incubation time post-transfection was therefore 48 hours. Cell movement was monitored using microscopy. Photographs were taken immediately and at 24 hours after scratching. The relative wound area was quantitatively evaluated using ImageJ software (National Institutes of Health).

Transwell assay

Transwell assay was employed to detect cell migration. Cells were transfected with siIDH2 or siCON in 6-well tissue culture plates for 24 hours, followed by transfer of 5×10⁵/mL cells in the upper transwell chamber (24 well plate) and culture with FBS free medium. Complete growth medium with 10% FBS was added to the lower chamber and incubated for another 24 hours. Then, cells on the upper side (nonmigrating cells) were removed and migrated cells on the lower face were washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI and counted on 5 random high-power fields (×200) under a microscope and averaged.

Human Tissue Resource Bank

Total of 59 invasive ductal breast cancer specimens were obtained from patients who underwent surgery from 2016 to 2019 in the Chungnam National University Hospital (Daejeon, Korea). Research protocols for this study were proved by Chungnam National University Hospital Institutional Review Board (IRB approval number: 2020-07-079). Written informed consent was obtained from all patients.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software (GraphPad Software). The Student t-test was used to evaluate the differences between two groups, one-way analysis of variance was used to evaluate multiple comparisons, and data are presented as mean ± standard error of the mean. A value of P<0.05 indicated statistical significance. All data in vitro experiments are representative of at least three independent experiments.

RESULTS

IDH isoform mRNA expression in different stages of human breast cancer tissues

Breast cancer has a high proportion of tumor stem cells, and requires a lot of energy through oxidation of the mitochondrial TCA cycle. α-KG, an intermediate product of the TCA cycle, can be used after conversion into glutamine and exported from the mitochondria as an essential amino acid for cancer cell metastasis and growth. We investigated IDH isoform messenger RNA (mRNA) expression in different stages of breast cancer tissues (stages 0–3) and normal breast epithelial tissues provided by the Human Resources Bank of Chungnam National University Hospital. Unlike IDH1 and IDH3, only IDH2 mRNA expression was increased in proportion to the breast carcinoma stage (Fig. 1A–C, Supplementary Fig. 1). Notably, the level of IDH2 mRNA in stages 2 and 3 breast cancer tissues was significantly elevated compared to normal tissue. The concentrations of α-KG in stages 1, 2, and 3 were also increased in a stage-dependent manner (Fig. 1D). Therefore, we confirmed elevated mRNA levels of IDH2 (stages 2–3) and α-KG in breast cancer patient tissues.

IDH isoform mRNA expression in different human breast cancer tissue types

Breast cancer is classified by stage and divided into four types based on estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. This includes triple positive, ER and PR positive, HER2 positive, and triple-negative. We grouped 59 breast cancer patient tissues according to the abovementioned types and measured IDH isoform mRNA expression by qPCR. IDH2 mRNA expression was increased in HER2 positive and triple-negative compared to the normal group (Fig. 2A–C). However, there was no significant difference in IDH1 and IDH3 mRNA and α-KG expression (Fig. 2D). These results indicated that the mRNA levels of IDH2 in the HER2 positive and triple-negative types showed higher expression than the normal group.

IDH isoform protein expression in different human breast cancer tissue types

As the IDH2 mRNA level was significantly elevated in stages 2 and 3, we selected stages 2–3 breast cancer patient tissues and confirmed protein expression levels of IDH1, IDH2, and IDH3A. Expression of IDH2 was greatly increased in all types of tissues (Fig. 3). Although IDH3A expression was also increased in the triple positive, ER and PR positive, and HER2 positive tissues, the difference was not statistically significant. These results suggest that IDH2 may be involved in the development of breast carcinoma.

IDH isoform protein expression in different breast cancer cell lines

We analyzed IDH1, IDH2, and IDH3A protein expression in cancer cell lines (triple positive, BT474; ER and PR positive and HER2 negative, MCF7; ER and PR positive and HER2 positive, SKBR3; triple negative, MDA-MB-231) and in normal breast epithelial cells (MCF10A) to determine whether IDH isoform expression changed. IDH2 expression was significantly elevated in MCF7, SKBR3, and MDA-MB-231 cells compared to MCF10A cells (Fig. 4). In addition, the level of IDH1 protein in MCF7 cells increased exceptionally, but there were no changes in BT474, SKBR3, and MDA-MB-231 cells. Although the IDH isoform protein expression was inconsistent with its mRNA expression in breast cancer tissues, IDH2 status was significantly associated with stages 2 and 3, HER2 positive, and triple-negative in patient tissues and cell lines. Additionally, the triple-negative type showed the highest increase compared to the other types.

IDH2 downregulation inhibits cell proliferation and migration by inducing cell cycle arrest in MDA-MB-231 cells

As IDH2 expression in triple-negative tissues and MDA-MB-231 (triple-negative) cells increased the most, we downregulated IDH2 by siRNA transfection of MDA-MB-231 cells to determine whether IDH2 deficiency exerted an anti-cancer effect in triple-negative breast cancer cells. IDH2 deficiency reduced cell proliferation (Fig. 5A). Moreover, transwell migration was also reduced in IDH2-deleted cells compared to control cells (Fig. 5B). Scratch wound coverage also showed reduced expression following IDH2 deletion (Fig. 5C). Downregulation of IDH2 arrested cells in the G0/G1 phase of the cell cycle (Fig. 5E) and was associated with increased cell cycle regulatory protein levels of p16 and p21 (Fig. 5D). These in vitro studies demonstrate that IDH2 deficiency significantly decreased proliferation and migration and showed an anti-cancer effect in MDA-MB-231 cells.

DISCUSSION

IDH is an essential metabolite in the TCA cycle, and its upregulation or mutation is associated with breast cancer progression and prognostics. This study analyzed the levels of three IDH isoforms and α-KG in 59 different breast cancer patient tissues at different stages. IDH2 was significantly upregulated in stages 2 and 3 breast cancer tissues and various breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231) associated with elevated α-KG expression. By contrast, IDH1 and IDH3 showed no significant differences between normal and breast cancer tissues. IDH2 knockdown inhibited proliferation and migration of MDA-MB-231 cells. Therefore, as a mitochondrial NADP-dependent enzyme, IDH2 is a potentially valuable target for treating human breast cancer.

Previous studies have investigated the possible roles of IDH isoforms in numerous malignancies and revealed that IDH mutations, upregulation, or repression can induce 2HG production, disturb antioxidant defense mechanisms, and regulate DNA methylation, which may contribute to cancer development [5,11,14]. Liu et al. [11] demonstrated significantly lower IDH1 expression in breast cancer than in adjacent normal tissues. Downregulation of IDH1 in breast cancers activated snail expression, which may be useful as an independent marker of breast cancer prognosis. IDH1 mutations are associated with various human cancer types, including breast cancer [14,15]. This study observed increased IDH1 expression in MCF-7 cells and found no differences in IDH1 between normal and breast cancer tissues. It is debatable whether IDH3 has a vital diagnostic and therapeutic value in cancer treatment. Al-Khallaf [5] suggested that the unidirectional structure of IDH3 is unlikely to induce reactive oxygen species formation and be carcinogenic. Conversely, IDH3A expression relates to poor postoperative survival rates in breast and lung cancer patients [16]. We found significant differences in IDH3 mRNA and protein expression between normal and breast cancer tissues, and therefore, IDH1 and IDH3 are not suitable as candidate markers for breast cancer prognosis.

However, IDH2 mutations and expression have attracted the most attention for their applications in cancer diagnosis and research. The IDH2 (R172) mutation has been detected in 77% of rare breast cancers (solid papillary carcinoma with reverse polarity), affecting canonical genes in the PI3K pathway and driving tumorigenesis. It is believed that the IDH2 mutation catalyzes α-KG directly into 2HG, which antagonizes α-KG-dependent dioxygenases and leads to alternations in cancer metabolism. Additionally, wild-type IDH2 expression was lower in the early phase but higher in the advanced phase of colon carcinoma [10]. Upregulated IDH2 expression was associated with a high risk of non-invasive and invasive breast cancer recurrence [12]. IDH2 did not change in stage 1 human breast cancer tissue in this study but was upregulated in stages 2 and 3 associated with increased α-KG expression, mainly in triple-negative and (ER−/PR−/HER+) patient tissues. However, although the IDH2 mRNA expression was meaningfully elevated in the HER2 positive and triple-negative subtypes, there was no significant increase in the α-KG levels (Fig. 2B and D). It is difficult to explain this discrepancy results which should be explored in the further research. Therefore, in these types of breast cancer, IDH2 status is more suitable as a biomarker. In vitro deletion of IDH2 in the triple-negative cell line MDA-MB-231 showed an anti-tumor effect by inhibiting cell proliferation and migration and arresting the cell cycle, but not autophagy (Supplementary Fig. 2). The underlying mechanisms of IDH2 in breast cancer remain to be clarified.

In conclusion, IDH2 was significantly upregulated in stage 3 breast cancer tissues and cell lines, essential for arresting the cell cycle and inducing breast cancer proliferation. This study suggests that IDH2 is a promising target for breast cancer treatment.

CONFLICT OF INTEREST

No potential conflict of interest relevant to this article was reported.

Notes

FUNDING

This work was supported by research fund of Chungnam National University.

SUPPLEMENTARY MATERIALS

Supplementary materials are available at the Korean Journal of Clinical Oncology website (http://www.kjco.org/).

kjco-19-2-60-Supplementary-Fig-1.pdf

kjco-19-2-60-Supplementary-Fig-2.pdf

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Fig. 1

IDH isoform mRNA expression in different stages of human breast cancer tissues. (A–C) mRNA expressions of IDH family (IDH1, IDH2, and IDH3A) were detected using a qPCR. β-Actin was used as an internal control. (D) Intracellular α-KG levels in different stages of human breast cancer tissues. These data are presented as mean ± standard error of the mean of different human samples. IDH, isocitrate dehydrogenase; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; α-KG, α-ketoglutarate; FC, fold change. ^a)P<0.05 versus normal. ^b)P<0.01 versus normal. ^c)P<0.0001 versus normal.

Fig. 2

IDH isoform mRNA expression in different human breast cancer tissue types. (A–C) mRNA expressions of IDH family (IDH1, IDH2, and IDH3A) were detected using a qPCR. β-Actin was used as an internal control. (D) Intracellular α-KG levels in different stages of human breast cancer tissues. These data are presented as mean ± standard error of the mean of different human samples. IDH, isocitrate dehydrogenase; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; α-KG, α-ketoglutarate; FC, fold change; PPP, triple positive; PPN, estrogen receptor and progesterone receptor positive; NNP, human epidermal growth factor receptor 2 positive; NNN, triple-negative. ^a)P<0.05 versus normal.

Fig. 3

IDH isoform protein expression in different human breast cancer tissue types. (A–D) Immunoblotting analysis of IDH family (IDH1, IDH2, and IDH3A) in tissues. β-Actin was used as internal control. Relative protein levels were quantified by densitometric analysis (n=4 patients in every group). These data are presented as mean ± standard error of the mean of different human samples. IDH, isocitrate dehydrogenase; PPP, triple positive; PPN, estrogen receptor and progesterone receptor positive; NNP, human epidermal growth factor receptor 2 positive; NNN, triple-negative. ^a)P<0.05 versus normal.

Fig. 4

The protein expression on IDH family in breast cancer cell lines. (A–D) Immunoblotting analysis of IDH family (IDH1, IDH2, and IDH3A) in several breast cancer cell lines. β-Actin was used as internal control. All experiments are representative of five independent experiments. These data are presented as mean ± standard error of the mean of five independent experiments. IDH, isocitrate dehydrogenase; PPP, triple positive; PPN, estrogen receptor and progesterone receptor positive; NNP, human epidermal growth factor receptor 2 positive; NNN, triple-negative. ^a)P<0.05 versus normal cell line (MCF10A). ^b)P<0.001 versus normal cell line (MCF10A).

Fig. 5

IDH2 deficiency suppresses cell proliferation and migration by inducing cell cycle arrest in MDA-MB-231 cells. MDA-MB-231 cells were transfected with siIDH2 (50 pmol), and incubated for 48 hours. (A) Cell proliferation was measured by CCK-8 kit. (B) Transwell assay was conducted to determine cell migration. Scale bar: 50 μm. (C) After transfection for 24 hours, MDA-MB-231 cells were wounded for another 24 hours. Images were obtained using a light microscope. (D) Immunoblotting analysis of cell cycle arrest marker p16 and p21. β-Actin was used as internal control. (E) Cell cycle arrest was detected using fluorescence-activated cell sorting (FACS) analysis. All experiments are representative of five independent experiments. These data are presented as mean ± standard error of the mean of five independent experiments. IDH, isocitrate dehydrogenase; siIDH2, small interfering IDH2 RNA. ^a)P<0.05 versus control cells. ^b)P<0.01 versus control cells. ^c)P<0.001 versus control cells.