https://www.mdpi.com/1422-0067/21/23/8982/htm

국립 암센타

교모세포종에 코미녹스가 약으로 효과적으로 활용 입증 논문 발표


Akt활성이 높은 교모세포에서 메타아세나이트나트륨의 종양방지효과 입증 논문 발표


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국립 암센타




Akt활성이 높은 교모세포에서 메타아세나이트나트륨의 종양방지효과

Anti-Tumor Effects of Sodium Meta-Arsenite in Glioblastoma Cells with Higher Akt Activities

by
Eun Jeong Lee
 1,†？,
Jee Young Sung
 1,†？,
Kyung Hee Koo
 1？,
Jong Bae Park
 2？,
Dae Hong Kim
 3？,
Jaegal Shim
 1？,
Chang Hoon Lee
 4？,
Jongsun Park
 5？ and
Yong-Nyun Kim
 1,*？

1

Division of Translational Science, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si 10408, Korea

2

Department of Cancer Biomedical Science, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si 10408, Korea

3

Division of Convergence Technology, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si 10408, Korea

4

College of Pharmacy, Dongguk University-Seoul, Goyang 10326, Korea

5

Department of Pharmacology, College of Medicine, Chungnam National University, Yuseong-gu, Daejeon 35015, Korea

*

Author to whom correspondence should be addressed.

†

These authors contributed equally.

Int. J. Mol. Sci. 2020, 21(23), 8982; https://doi.org/10.3390/ijms21238982

Received: 28 October 2020 / Revised: 23 November 2020 / Accepted: 24 November 2020 / Published: 26 November 2020

(This article belongs to the Special Issue Molecular Connection and Influence of Extracellular Matrix with Cancer)

Abstract

Glioblastoma is a type of aggressive brain tumor that grows very fast and evades surrounding normal brain, lead to treatment failure. Glioblastomas are associated with Akt activation due to somatic alterations in PI3 kinase/Akt pathway and/or PTEN tumor suppressor. Sodium meta-arsenite, KML001 is an orally bioavailable, water-soluble, and trivalent arsenical and it shows antitumoral effects in several solid tumor cells via inhibiting oncogenic signaling, including Akt and MAPK. Here, we evaluated the effect of sodium meta-arsenite, KML001, on the growth of human glioblastoma cell lines with different PTEN expression status and Akt activation, including PTEN-deficient cells (U87-MG and U251) and PTEN-positive cells (LN229). The growth-inhibitory effect of KML001 was stronger in U87-MG and U251 cells, which exhibited higher Akt activity than LN229 cells. KML001 deactivated Akt and decreased its protein levels via proteasomal degradation in U87-MG cells. KML001 upregulated mutant PTEN levels via inhibition of its proteasomal degradation. KML001 inhibited cell growth more effectively in active Akt-overexpressing LN229 cells than in mock-expressing LN229 cells. Consistent with these results, KML001 sensitized PTEN-deficient cells more strongly to growth inhibition than it did PTEN-positive cells in prostate and breast cancer cell lines. Finally, we illustrated in vivo anti-tumor effects of KML001 using an intracranial xenograft mouse model. These results suggest that KML001 could be an effective chemotherapeutic drug for the treatment of glioblastoma cancer patients with higher Akt activity and PTEN loss.
 
Keywords:   glioblastoma; PTEN; Akt; sodium meta-arsenite
1. Introduction

Glioblastomas are intra-axial tumors that originate from neuroglial cells of the central nervous system (CNS) [1,2]. Glioblastomas are known to display cellular heterogeneity with stem-like glioblastoma stem cells at the apex [3]. Glioblastoma multiforme (GBM) is the most common and aggressive malignant primary brain tumor in adults and is a major cause of morbidity and mortality among neurosurgical patients, with only 12% surviving beyond 36 months (long-term survivors) [4]. The current standard treatment strategy for glioblastoma is multimodal, involving maximal surgical resection followed by radiotherapy with concomitant and adjuvant temozolomide (TMZ) [2]. Although therapeutic advances in diagnosis have been made, glioblastoma is resistant to current treatments, such as chemotherapy and radiation, and effective treatment options are lacking. Recent insights into the biology of glioblastoma, including those from The Cancer Genome Atlas, have revealed important genetic events in human glioblastomas, such as gene amplification, mutation, and deletion [1,5]. The most frequent alterations in gliomas include dysregulation of growth factor signaling via amplification and mutational activation of receptor tyrosine kinase genes such as EGFR. Phosphatidylinositol-3-kinase (PI3K) signaling pathways are also activated due to genetic alteration in the phosphatase and tensin homolog (PTEN) tumor suppressor gene on 10q23.3 at the level of loss of heterozygosity, mutation, and methylation in at least 60% of gliomas [6].

PTEN, a tumor suppressor, is a lipid phosphatase that hydrolyses phosphate in position 3′ from phosphoinositide, thereby opposing mitogenic signaling mediated by PI3K [7]. The loss of PTEN function via its mutation or deletion leads to hyperactivation of PI3K signaling [7]. The loss of PTEN function has been linked to tumor malignancy, including metastasis and resistance to radiotherapy and chemotherapy in brain and breast cancer patients [8,9,10]. The major downstream effector of PI3K signaling is the pro-survival serine/threonine kinase, Akt [7]. Akt is activated by phosphorylation at Thr 308 (T308) by PDK1 and is fully activated by further phosphorylation at Ser 473 (S473) by mTORC2 [11]. Activated Akt phosphorylates numbers of substrates, and the majority of these substrates are involved in cell survival, proliferation, and metabolism. Uncontrolled Akt activation occurs through either overexpression and mutation, a loss of negative regulator PTEN, or the activation of PI3K pathways. These outcomes have been reported in a number of cancers including glioblastoma, breast, and prostate cancers [1,7,12]. Therefore, Akt is an attractive target for the control of cancers where the PI3K？Akt pathway is upregulated, such as glioblastoma.

Arsenic-based drugs have been used for centuries. Arsenic trioxide (As2O3), an arsenic derivative, exerts potent anti-tumor effects in vitro and in vivo [13]. As2O3 is known to be an effective inducer of apoptosis in patients with relapsed acute promyelocytic leukemia (APL) [13,14]. Because it also has a potent effect in vitro against different types of malignant leukemia as well as APL, there is increasing interest in the therapeutic development of arsenic compounds for the treatment of various solid tumors. Arsenic compounds are known to induce cell death via multiple pathways, such as reactive oxygen species (ROS) generation, JNK activation, and the activation of pro-apoptotic proteins such as Bax [13,15]. Recently, As2O3 was reported to decrease Akt protein levels in a caspase-dependent manner [16]. Akt is highly activated in glioblastomas when PTEN function is lost, so it is possible that an arsenic compound could control glioblastomas via Akt downregulation. As2O3 is poorly water-soluble and it must be dissolved with sodium hydroxide and then adjusted to physiologic pH, yielding s odium meta-arsenite (NaAsO2) [17]. NaAsO2, KML001, is a water soluble, and thus orally bioavailable arsenic compound that has been reported to have anti-tumor effects on several tumors, including prostate cancer and glioma cells [17,18]. KML001 is known to exerts its cytotoxic effects via its binding to telomeric sequences, which leads to shortened telomeres in prostate cancer [17,18]. KML001 has anti-tumoral effects on non-Hodgkin lymphoma cells via inhibiting cell signaling including PI3K/Akt and MAPK [13,14]. Recently, KML001 exhibits anti-tumoral effects in multiple myeloma cells via Akt inactivation and PTEN activation [19]. In addition, KML001 has entered Phase I/II clinical trials for treatment of prostate cancer [20] and Phase I clinical trials for advanced non-small-cell lung cancer as well as other platinum-responsive malignancies (https://clinicaltrials.gov). Because KML001 induces Akt inactivation and PTEN activation [19] and because Akt activation with PTEN mutation is associated with glioblastoma, in this study, we evaluated the anti-tumor effects of KML001 using PTEN-negative human glioblastoma U87-MG cells in vitro and in vivo, and investigated the possible mechanisms involved in this process.

2. Results

2.1. Expression Levels of PTEN and Akt Activation in Human Glioma Cell Lines

First, we examined the level of PTEN expression and Akt activation in several glioma cell lines, including U87-MG, LN229 and U251 cells. As shown in Figure 1A, when PTEN mRNA levels were evaluated by RT-PCR, PTEN expression was undetectable in U251 cells, but both U87-MG and LN229 cells expressed comparable levels of PTEN mRNA. However, protein levels of PTEN were only detected in LN229 cells (Figure 1B). U87-MG cells are known to contain in-frame deletion of Exon 3 within the tensin region [21]. Although comparable levels of this mutation were expressed at the transcriptional level, the mutant PTEN protein was not detected by immunoblotting in U87-MG cells (Figure 1B left panel and lower panel). Akt activation was higher in U87-MG and U251 cells with little PTEN expression, as assessed by immunoblotting using anti-phospho-Akt (pAkt), although Akt expression was higher in LN229 cells (Figure 1B). However, longer exposure of immunoblot could recapitulate mutant PTEN protein in U87 cells (Figure 1B, right panel). This mutation may be critical for PTEN turnover, or the antibody might not recognize this mutant protein in the same way as it does the wild-type PTEN. When cell growth rates were compared, U87-MG cells grew faster than U251 and LN229 cells (Figure 1C). These data indicate that PTEN downregulation was positively correlated with elevated Akt activation in the glioma cell lines tested in this study.

Ijms 21 08982 g001 550 

Figure 1. Expression levels of PTEN and Akt and cell growth in human glioblastoma cell lines. (A) Total RNA was extracted from glioma cell lines as indicated and subjected to RT-PCR to measure PTEN mRNA levels. (B) Cells were lysed and 30 μg of total cellular proteins was analyzed by immunoblotting using anti-PTEN, -pAkt, -Akt, and β-actin antibodies. β-actin was used as a loading control. In the right panel, film was exposed a little longer for PTEN immunoblot. The levels of proteins were quantified by a densitometry and normalized to the loading control β-actin (lower panel). (C) 1 × 105 cells were seeded and were grown in the culture media condition for 4 days. The cell numbers of each cell line were counted after trypan blue-staining at indicated times. Statistical analysis was conducted using parametric one-way ANOVA test and post hoc test (Bonferroni correction). Error bars represent standard deviations of the mean of three measurements (* p < 0.05, ** p < 0.01). These experiments were performed three independent times with comparable results.

2.2. Effects of KML001 on Glioma Cell Growth and Akt Activity

To test whether KML001 could induce cell growth inhibition in glioma cells, glioma cells were treated with various concentrations of KML001 for 24 h in either serum-free medium or 10% FBS-containing medium. Treatment of KML001 resulted in a dose-dependent cell growth inhibition, regardless of serum presence, in all the cell lines tested in our study (Figure 2A,B, and Supplementary Figure S1). Compared with other cell lines, U87-MG cells, which express a mutant PTEN and possess higher Akt activity, showed higher sensitivity to lower doses of KML001. U251 cells are PTEN-negative and were also notably responsive to KML001. LN229 cells were the least responsive to KML001. To test whether KML001-induced growth inhibition was due to cell death, we carried out a trypan blue dye exclusion assay after KML001 treatment. Cell death increased in a dose-dependent manner in all three cell lines, and U87-MG cells showed greater cell death after KML001 treatment than the other two cell lines (Figure 2C). Akt activation is frequent in glioma because PI3K？Akt pathways are often activated by either growth factor receptor signaling or loss of function of PTEN [6,22]. Activated Akt exerts its pro-survival activity by phosphorylating various proteins for survival signaling. As the cell lines we used expressed different levels of pAkt, we tested whether KML001 affected Akt activity. Because 10 μM KML001 inhibited cell growth of U87-MG cells significantly when compared with other cell lines, we used 10 μM KML001 to examine changes of Akt activity in three cell lines. Upon 10 μM KML001 treatment, Akt phosphorylation and Akt protein levels decreased in U87-MG and U251 cells, whereas both pAkt and Akt protein levels increased in LN229 cells in same dose of KML001, which were the least responsive to KML001 (Figure 2D,E). Interestingly, there were detectable levels of p53 expressed in LN229 and U251 cells initially but there was little p53 expression in U87-MG cells. However, p53 was dramatically upregulated following KML001 treatment in the U87-MG cells but not in U251 or LN229 cells (Figure 2D,E). U87-MG cells are known to be PTEN-deficient [23] and we could not detect PTEN at the protein level, although its mRNA levels were notable (Figure 1A,B). However, KML001 increased the PTEN level in U87-MG cells, which exhibited non-detectable levels of PTEN initially (Figure 2D). These findings indicate that KML001 induced growth inhibition depending on Akt activity and PTEN expression status.

4.14. Immunofluorescence Analysis

Cells were fixed with 1% paraformaldehyde in PBS at 4 °C for 20 min, permeabilized and blocked in 3% BSA in PBS for 1 h at room temperature. Cells were then incubated with anti-p53 antibody for 4 °C overnight, and then incubated with Alexa 488-conjugated antibody for 1 h at room temperature. For nucleus staining, cells were incubated with DAPI in PBS. Cells were examined using confocal microscopy (Zeiss, Jena, Germany).

4.15. Statistical Analysis

Comparison between two groups were performed using Student’s t-test. Multiple group comparisons were made parametric one-way ANOVA followed post hoc test (Bonferroni correction). Data represent average values and standard deviations (error bars) obtained from three independent experiments.

5. Conclusions

Glioblastoma is frequently associated with PTEN loss or mutation, which leads to AKT activation. KML001, sodium arsenite induced growth inhibition due to apoptosis of glioma cell lines more sensitively in PTEN-deficient cells with high-AKT activity than in PTEN-wild type cells. KML001 inactivates AKT via proteasomal degradation of AKT. Additionally, in vivo anti-tumor effects of KML001 was observed in orthotopically xenografted model. These data indicate that KML001 could be an effective chemotherapeutic drug depending on higher Akt activity due to PTEN deletion/mutation in glioblastoma cancer patients.

Supplementary Materials

The following are available online at https://www.mdpi.com/1422-0067/21/23/8982/s1.
4.14. 면역 형광 분석

세포는 4 °C에서 PBS에서 1% 파라포름알데히드로 20분 동안 고정되었고, 투과성 BSA에서 1 Hat 실온에서 3% BSA로 차단되었다. 그리고 나서 세포들은 하룻밤 사이에 4 °C의 항 p53 항체로 배양되었고, 그리고 나서 1 온도의 실온에서 알렉사 488-결합 항체와 함께 배양되었다. 핵 얼룩의 경우, 세포는 PBS에서 DAPI와 함께 배양되었다. 세포는 공초점 현미경을 사용하여 검사되었습니다(Zeiss, Jena, Germany


4.15. 통계적 분석

두 그룹 간의 비교는 학생의 t-검정을 사용하여 수행되었습니다. 다중 그룹 비교는 사후 검정에 따라 모수 일원 분산 분석(Bonferroni correction)이 수행되었습니다. 데이터는 세 가지 독립 실험에서 얻은 평균 값과 표준 편차(오류 막대)를 나타냅니다.


5. 결론

교모세포종은 종종 PTEN 손실이나 돌연변이와 연관되어 AKT 활성화로 이어진다. KML001, 나트륨 비소산나트륨은 PTEN-wild type cells보다 높은 AKT 활성을 가진 PTEN 결핍 세포에서 교모 세포 라인의 선세포화로 인해 성장 억제를 더 민감하게 유도했다. KML001은 AKT의 단백질의 저하를 통해 AKT를 비활성화합니다. 또한 KML001의 체내 종양 방지 효과는 정형외대증 모델에서 관찰되었습니다. 이러한 데이터는 KML001이 교모세포종 암 환자의 PTEN 삭제/변조로 인해 Akt 활동에 따라 효과적인 화학 치료제가 될 수 있음을 나타낸다.

자금 지원 : 이 연구는 국립암센터, 한국(1810082 및 1810931) 및 한국보건기술연구개발부(HI17C0792)의 연구 보조금으로 지원되었다.

양회장은 논문으로 입증 발표 하겠다고 했습니다

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