Indirubin in Vitro Apoptotic Effect Towards Chronic Lymphocytic Leukaemia (CLL) Lineage

doi:10.21203/rs.3.rs-3775875/v1

Download PDF

Research Article

Indirubin in Vitro Apoptotic Effect Towards Chronic Lymphocytic Leukaemia (CLL) Lineage

https://doi.org/10.21203/rs.3.rs-3775875/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Chronic lymphocytic leukemia (CLL) is a chronic condition that usually affects elderly people. The etiology is unknown, however the current hypothesis is that over time the haematopoietic stem cells may acquire mutations that will lead to irregularity in apoptotic mechanism (e.g. BCL2 mutation). Dangui Luhui Wan consist in mix of 11 herbs used by the Chinese Medicine. This herbal compound has proven to have antitumoral activities on various types of cancer cells. A derivate from Dangui Luhui Wan is indirubin-3'-monoxime (I3M). This substance act as selective inhibitor of cyclin-dependent kinases (CDKs) and can induce cell apoptosis. The aim of this study was to test the efficacy of I3M against CLL cells in vitro. We evaluated the expression of apoptotic proteins Bcl2/Bax and CDK1/2 using real-time PCR. Peripheral mononuclear cells (PBMCs) obtained from 14 patients were treated with 20 μM of I3M for 48hrs. After treatment a reduced Bcl-2 expression was noticed. No significant changes were seen for Bax. However, an increased Bax/Bcl-2 ratio was documented, suggesting that mitochondrial pathway is involved in I3M apoptotic-mechanism of action. Interestingly, I3M could inhibit the expression of CDK2, while it does not affect the expression of CDK1. These results indicated that I3M exerts anti-tumor effects through induction apoptosis and inhibition of CDK2. Further studies are now needed to clarify the exact mechanism of action of I3M in CLL and possibly in other tumoral cell lines.

Chronic lymphocytic leukemia

CLL

indirubin

Bcl2CDK1

CDK2

B-Chronic lymphocytic leukemia (B-CLL) CLL accounts for 25–30% of leukemias in Western countries [1]. CLL has a higher incidence among men (M/F = 1.7/1) [2]. Classical presentation includes marked lymphocytosis in the peripheral blood. Bone marrow involvement is almost always seen [3]. A bone marrow biopsy is not an obligatory test for establishing CLL diagnosis. However, bone marrow biopsy may be useful for assessing the level of response to treatment and/or follow up. While the pathogenesis of the disease remains largely in debate, a growing body of evidence has focused on the aberrant regulation of apoptotic mediators such as B-cell lymphoma 2 (Bcl-2) family members of anti-apoptotic (e.g., Bcl-2, Bcl-xl, Bcl-w, and Mcl-1) and pro-apoptotic (e.g., Bax, Bak, and Bok) proteins caused by genetic mutations [4]. Interestingly, the overexpression of Bcl-2 increases the Bcl-2/Bax ratio, which inhibits the caspase-dependent apoptosis of CLL cells and reduces the efficacy to immune- and chemotherapy[5].New treatments targeting these mechanisms have recently become available [6]. Previous studies have demonstrated that the alteration of cell cycle regulatory genes, may contribute to clonal expansion of malignant B cells [7, 8]. Cyclin-dependent kinases (CDKs) are key regulators of cell cycle, transcription and apoptosis [9]. Perturbation of CDKs or their related cyclins encoding genes expression is frequently associated with tumor formation [10, 11]. A transmembrane protein called the B cell receptor (BCR) is located on the surface of a B cell. A membrane-bound immunoglobulin molecule and a signal transduction moiety make up a B cell receptor [12]. Translocation and overexpression of cyclin D1 and cyclin D2 have been reported in CLL [13]. Cyclin D2 has a key role in CD5 B cell proliferation and its action is carried out through BCR. BCR consist of progression into G1 cell cycle phase in B lymphocytes is controlled by components of the cell cycle. CyclinD2 plays a key role in cell cycle regulation [14, 15].

Targeting CDK1 might be effective for the treatment of hematologic malignancies due to its ability to phosphorylate retinoblastoma protein (pRb) upon additionally binding to cyclin D [16].. CDK1 is involved in cell cycle through G2 and M phases and regulates important cellular processes such as: DNA damage repair, mesenchymal stem cell (MSC) differentiation and epigenetic gene silencing [16, 17]. Although CDK1 and CDK2 exert an inhibitory action on cancer cell invasion, the association of CDK1 with disease progression has been demonstrated in acute myeloid leukaemia (AML) and chronic myelomonocytic leukemia (CMMoL [18, 19]. A higher expression of CDK1 has been observed in breast cancer, gliomas, and colon cancer. An increased expression of CDK2 has been reported in breast cancer [13]. Several CDKs inhibitors have demonstrated to carrier an anti-proliferative and pro-apoptotic effect including CDKI-73© (a non-selective CDK9 inhibitor), Flavopiridol© (a pan CDK inhibitor targeting CDKs 1, 2, 4, 6, 7, and 9), Dinaciclib© (inhibiting CDKs 1, 2, 5, and 9), and other pan CDK inhibitors [20]. Furthermore, CDKs inhibitors induce apoptosis by damaging mitochondria in human leukemia cells. CDK1 modulation may therefore represent a novel therapeutic approach for CLL. However, CDKs inhibitors have severe side effects such as tumor lysis syndrome, secretory diarrhea, and other [21]. and their clinical use should be performed by experienced haematologists.

Besides using common and new therapeutic approaches in CLL, herbal medicine deserves to be also considered.

Dangui Luhui Wan is made up of 11 herbs and is quite a popular drug of the Chinese Medicine. Indirubin-3-monoxime (I3M) is an active ingredient of Dangui Luhui Wan. I3M is isolated from plant species, including Baphicacanthus cusia, Indigofera suffruticosa and Polygonum tinctorium [22]. I3M has been showed to have anticancer effects in several types of solid cancers and hematologic malignancies such as human glioma cancer [23], human ovarian cancer [24], and acute lymphoblastic leukemia (ALL) [25]. Interestingly, the anticancer mechanisms of I3M is believed to be sustained by its inhibitory action on cyclin-dependent kinases (CDKs). I3M will compete with ATP binding at the catalytic site, inducing apoptosis and autophagy, and suppressing STAT3 signaling [24, 25]. To the best of our knowledge, no previous studies have been carried using I3M for CLL. In this study, we investigated the effects of I3M on cell survival, including expression levels of CDK1/CDK2 and Bcl-2/ Bax, apoptosis and proliferation of cells in CLL.

2.1. Subjects

A total of 14 newly diagnosed CLL patients with a mean age of 60 ± 4 years were selected according to the iWCLL criteria [26]. An age and gender matched control group (n = 8) was selected for this study. The main demographic characteristics of patients are summarized in Table 1. This study was approved by the Ethical Committee of Semnan University of Medical Sciences, (241; 1395.01.15). All patients and controls were asked to sign an informed consent before sampling.

Table 1

Demographic and laboratory characteristics of patients and Healthy subjects .
Patients (CLL)	1	2	3	4	5	6	7	8	9	11	12	13	14	Healthy subjects (ranges)
Age	61	64	60	61	63	62	64	60	62	64	61	63	64	60–66
Gender	M	M	M	F	M	M	F	F	F	M	M	M	M	NA
RAI stage	0	0	0	0	0	0	0	0	0	0	0	0	0	NA
Binet stage	A	A	A	A	A	A	A	A	A	A	A	A	A	NA
WBC/µL	32000	30000	40000	35000	28000	50000	35000	42000	25000	60000	25000	40000	36000	5000–7000
Lymphocytes/ µL	14320	20350	15320	11500	8050	6250	3150	3140	47100	22360	7350	8600	45450	450–9640
Haemoglobin (g/dL)	14.3	12.2	16.6	15.1	14.5	12.4	14.8	15.4	16.3	15.2	14.3	16.3	14.3	12.2–14.3
Platelets (10³/ µL)	236	250	210	350	365	400	250	241	230	340	360	380	410	210–430
Beta 2 microglobulin (mg/dL)	2.0	2.5	2.8	2.3	3.2	2.9	2.1	2.4	2.7	3.7	4.1	3.6	4.2	< 2.5

2.2. Peripheral blood mononuclear cell (PBMC) isolation and cell culture

Mononuclear cells from CLL patients and healthy donors were isolated from fresh lithium-heparinized blood samples using Ficoll-Hypaque density gradient (Sigma©) and stored in Rnase and Dnase free tubes. Briefly, 10 ml phosphate buffer saline (PBS)-diluted peripheral blood samples were layered over the 5 ml of Ficoll–Hypaque. The tubes were centrifuged for 20 min at 500 G and PBMC were isolated from the interlayer and washed twice with PBS, and subsequently cultured in three different groups: i) treated with indirubin, ii) untreated (control group), and iii) treated with fludarabine (positive control).

The medium used was the same for all 3 groups and consisted of: RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin G and 100 mg/ml streptomycin (Sigma). Samples were incubated at 37°C in a humidified incubator with 5% CO2 24 h, 48 h and 72 h.

2.3. Cell viability assay

To determine the optimal concentration of I3M with the most anti-tumor effects and minimum cytotoxicity on normal cells, MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 5 mg/ml, Sigma) assay was used. To determine the optimal dose of I3M, 10⁵ PBMCs/well were seeded in 96-well plates and treated with different concentrations of I3M (0.1, 0.25, 0.5, 1, 5, 10, 20, 40, and 80 µM), and incubated for 24, 48, and 72 hrs. Subsequently, 10 µl/well of MTT solution (0.5 mg/ml) was added and cells were incubated for 4 h in 37°C. After adding 100 µl/well of MTT solvent (SDS 10%), optical density of each well was read at 570 nm by a plate reader (Stat Fax 2100 Microplate). Results were compared with untreated control cells. The group of cells treated with fludarabine was used as positive control. Data are expressed as mean ± SD of at least three independent experiments. Subsequently an apoptosis assay was performed for PBMCs and CD19 + cells cultured in different concentrations of I3M at 24, 48, and 72 h. According to the assay results, I3M concentration at 20 µM had the maximum cytotoxic antitumoral effect with lowest side effect on normal cells. I3M concentration at 20 µM was used on other cells of patients and healthy donors for the subsequent experiments such as MTT and Annexin V assay at 48 hrs. Data are expressed as the means ± SD of at least three independent experiments.

2.3. Apoptosis determination assay

Flowcytometry was used for assessing apoptosis induced by I3M. In brief, 3 × 10⁵ PBMCs/ well of patients with CLL and healthy subjects were cultured in 48-well plates supplemented with 20 µM of I3M and incubated for 48 hrs in 5% CO2 at 37°C. Annexin V/7-AAD kit (Biolegend©) was used for the assessment of apoptosis. This was performed according to the manufacturer’s instructions in PBMCs and CD19⁺ cell (anti-CD19-PE purchased from Biolegend). Subsequent results were quantified by Partec cyflow© (Partec©). All data were analyzed by FlowJo© software (Tree Star Inc©).

2.4. RNA extraction, cDNA synthesis, and qRT-PCR

To determine the impact of I3M on Bcl-2, Bax, CDK1, and CDK2 expression levels in CLL cells, 35 × 10⁵ PBMCs/well were cultured in the presence of I3M (20 Μm) in 12-well plates for 48 h. Subsequently, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific©) according to the manufacturer’s instructions. The quality and quantity of the extracted RNA were assessed through gel electrophoresis and Nano-Drop instrument (Thermo Fisher Scientific©), respectively. cDNA synthesis was conducted from 1µg of total extracted RNA using cDNA Synthesis Kit (High-Capacity cDNA Reverse Transcription Kit). Real-time qRT-PCR amplifications were performed using the SYBR Green Super mix (Ampilicon©).

The primer sequences included as following; Bcl-2 (sense): 5’-TCG GTGGGGTCATGTGTGTGG-3’ and antisense: 5’-CACAAAGGCATCCCAGCCTCC-3’; Bax (sense): 5’-CAAGCGCATCGGGGACGAACT-3’ and antisense: 5’-CTTGGTGCACAGGGCCTTGAG-3’; CDK1 (sense): 5’-CTTTGTGGCCCTCAAGAGTT-3’ and antisense: 5’-AGAGGCTTTCGACGAAACATC-3’; and the primer sequences for CDK2 (sense): 5’-AATCCGCCTGGACACTGAGAC-3’ and antisense: 5’-GTGAGAGCAGAGGCATCCATG-3’.

The gene expression of Bcl-2, Bax, CDK1, and CDK2 were normalized by RPLP0 as housekeeping internal controls. All experiments were performed by ABI 7900 HT©, and the 2^−ΔΔCt method was used to calculate the expression of each mRNA.

2.5. Statistical analysis

All statistical calculations were conducted with Prism 7.0.3 software (GraphPad Software©). After assessment of normality, Mann–Whitney U test was used to nonparametric comparisons and independent t-test was used to parametric data evaluation. Results are expressed as the mean ± standard deviation and a p < 0.05 was regarded as significant.

3.1. Determination of optimal concentration of I3M

The cytotoxicity percentage of I3M was investigated using MTT assay in CLL patients and healthy donors with different concentration: 0.1 to 80 µM for 24, 48 and 72 hours. As shown in Figure 1A, 1B and 1C. Fludarabine used as positive control.

3.2. Cytotoxic effect of I3M on CLL cells but not on healthy control cells

The results demonstrated that I3M used at 20 µM concentration had the highest cytotoxicity after incubation for 48 hours compared to controls (p= 0.005). Fludarabine as positive control had statistically significant p-value compared to controls (p= 0.0003). As shown in Figure 2B, statistically significant increase of cell cytotoxicity was observed following incubation with 20 μM of I3M for 48 h In CLL cells (n=14) (p=0.0001). The assessment of anti-proliferative/cytotoxic impact of I3M (20 μM) on PBMCs obtained from CLL patients, in comparison with untreated cells showed a statistically significant decrease in cell viability (p= 0.0001) (Figure 2A). However, no statistically significant difference in cell viability between I3M- treated group and fludarabine treated group was seen (P>0.5 Figure 2A). In contrast, no marked anti-proliferative/cytotoxic effect was observed in PBMCs of healthy controls upon treatment with I3M (20 μM), compared with untreated cells (p >0.5).

3.3. Apoptotic effect of I3M on PBMCs and CD19+ CLL cells

Apoptotic effects of I3M were determined on PBMCs and CD19+ CLL cells of CLL patients and healthy controls. Cells were cultured upon different concentrations of I3M including 0.1, 0.25, 0.5, 1, 5, 10, 20, 40, and 80 μM in different incubation times from 24, 48, to 72 hand the then assessed by flow cytometry analysis. The greatest apoptotic effect of I3M was seen with in concentration of 20 μM after 48 h of incubation (p= 0.0008; Figure 3).

As shown in Figure 4A, I3M (20 μM) induced apoptosis in PBMCs of CLL patients with CLL, in comparison with untreated cells. This was similar to the fludarabine apoptotic effect (p= 0.0001). However, I3M could not induce significant apoptosis in the cell growth with fludarabine and in those from healthy controls (p >0.5, Figure 4B).

3.4. Apoptotic impact of I3M on Bcl-2 expression

To explore the underlying mechanism of apoptosis in CLL induced by I3M, the expression levels of Bcl-2 and Bax were evaluated in I3M (20 μM)- treated PBMCs of CLL patients for 48 h in comparison to untreated cells. As presented in Figure 5A, although I3M could significantly reduce the expression of Bcl-2 in PBMCs of CLL patients (p= 0.03), there was no marked difference of Bax expression in I3M- treated cells, compared with untreated cells (p >0.5).

According to the Figure 5B the results also showed no statistically significant difference in expression of Bcl-2 and Bax expression levels between I3M (20 μM) treated and untreated PBMCs of healthy controls (p >0.5 respectively).

3.5. Inhibition of CDK2 expression through I3M treatment in CLL

To further test whether I3M can affect cell-cycle events, we evaluated the expression of CDK1 and CDK2 levels upon treatment of PBMCs obtained from CLL patients and healthy subjects with I3M (20 μM) for 48 hrs. This was compared to untreated cells. The expression of CDK1 levels had no statistically significant difference between I3M- treated cells and untreated cells of CLL patients and healthy controls as showed in figure 6A, whereas the levels of CDK2 showed a marked decreased expression upon treatment with I3M, compared to what seen in the untreated cells (p=0.03). However, no statistically significant difference was observed in the expression of CDK2 levels in PBMCs of healthy controls, compared with untreated cells (p >0.5) as showed in figure 6B.

With increasing development in pharmaceutical technology, discovery of active agents from the herbal medicine provides a new perspective for cancer treatment. These compounds are known to have some therapeutic effects, however rarely the same have been tested in scientifically studies. Indirubin-3ʹ -monoxime (I3M) is described to be a potent inducer of apoptotic cell death in a wide spectrum of human cancer cells with few cytotoxicity on normal cells, including breast cancer, acute lymphoblastic leukemia, chronic myelomonocytic leukemia, osteosarcoma, and neuroblastoma [27–29]. However, the exact mechanisms underlying the apoptotic cell death induced by I3M have not been fully elucidated. In this study, we provided convincing evidence that I3M-induces apoptosis through modulating the expression of anti-apoptotic Bcl-2 and also decreasing CDK2 expression.

Our first endpoint was aimed to find the best I3M concentration with the strongest anti-cancer effects and at the same time with the minimal toxicity on normal cells. This was done using different concentration of I3M (ranging between 0.1 µM to 80 µM). In line with previous studies, we shown that 20 µM of I3M is considered as the optimum concentration [30, 31]. Subsequent assessments were conducted using 20 µM of I3M. In accordance with previous reports, we provided evidences that I3M exerts apoptotic effects on the CLL cells by decreasing Bcl-2 expression [23, 27]. An increase in Bax expression was reported by Li et al. in glioma cells [23]. Similarly, Zhang et al. observed that I3M treatment led to the down regulation of Bcl-2, Bcl-xl, Mcl-1, survivin and upregulation of Bax in osteosarcoma cells [27]. Conversely, the expression level of Bax was not statistically different upon I3M exposure, compared with untreated cells. Treatment with I3M induces caspase-8 activation resulted in Bid cleavage, followed by Bax conformational change, cytochrome c release and cell apoptosis[32]. Previous studies shown that I3M-induced apoptosis in several cancer cells with the engagement of both the anti-apoptotic and pro-apoptotic Bcl-2 family members at the site of mitochondria [18, 24], which needed further investigations in the case of CLL.

Moreover, it was demonstrated that I3M act as inhibitor of CDKs and induce arrest in G2/M as well as in G1/S which mainly followed by apoptosis. Indirubin derivatives directly can bind to the ATP binding site of CDKs in an ATP-competitive manner and block the cell cycle [25]. Our results in CLL cells showed that I3M inhibited the expression of CDK2, while had no significant impact on CDK1 expression. These results implied that the down-expression of CDK2 contributed to the CLL cell cycle arrest. Identification of CDKs as specific molecular targets of I3M was first reported by the group of Meijer et al. [33] and followed by Marko et al. These authors showed the inhibitory impact of I3M on proliferation of mammary carcinoma cell line (MCF-7), by arresting the cells in the G2/M phase of the cell cycle, possibly through inhibiting CDK1[34]. Similarly, Lio et al. demonstrated that I3M reduced the expression of CDK2 and cyclin E, leading to cell cycle arrest at the G0/G1 phase in human neuroblastoma cell line (LA-N-1) [35].

In conclusion, our findings indicated that the anti-tumor effects of I3M in CLL cells might be mediated by suppressing anti-apoptotic proteins such as Bcl2 and inhibiting CDK2. Moreover, I3M exerted minimal cytotoxicity towards the normal cells. Better further characterization of the anti-tumor mechanisms of I3M is needed to support its role as a potential drug for the clinical treatment of CLL.

Author contributions:

All the authors of this manuscript participated in all stages of the project, including research design, execution of lab experiments, data collection and analysis, and manuscript writing.

Conflict of Interest:

The authors declare that there is no conflict of interests.

Ethical approval: All procedures performed in this study involving human participants were in accordance with the ethical standards of the Semnan University of Medical Sciences. This study was compiled and approved by the ethics committee of the Semnan University of Medical Sciences in compliance with the requirements (Code No: 241; 1395.01.15).and written informed consent was obtained from all subjects before participating in the study.

Consent to participate: All authors have read and approved the final version of the manuscript for publication.

Ahmed R, Osman IM. Clinical and haematological pattern of chronic lymphocytic leukaemia in sudanese patients. Int Blood Res Rev. 2017;7(1):1-10.
Hasan KM. Clinical aspects, immunophenotypic analysis and survival rate of chronic lymphocytic leukaemia patients in Erbil City, Iraq. Sultan Qaboos University Medical Journal. 2018;18(4):e461.
Spring LM, Zangardi ML, Moy B, Bardia A. Clinical management of potential toxicities and drug interactions related to cyclin‐dependent kinase 4/6 inhibitors in breast cancer: practical considerations and recommendations. The oncologist. 2017;22(9):1039-48.
Chiorazzi N, Rai KR, Ferrarini M. Chronic lymphocytic leukemia. N Engl J Med. 2005;352(8):804-15.
Fu C, Gong Y, Shi X, Sun Z, Niu M, Sang W, et al. Plumbagin reduces chronic lymphocytic leukemia cell survival by downregulation of Bcl-2 but upregulation of the Bax protein level. Oncol Rep. 2016;36(3):1605-11.
Hallek M, Al‐Sawaf O. Chronic lymphocytic leukemia: 2022 update on diagnostic and therapeutic procedures. American journal of hematology. 2021;96(12):1679-705.
Otto T, Sicinski P. Cell cycle proteins as promising targets in cancer therapy. Nature Reviews Cancer. 2017;17(2):93-115.
Sobecki M, Mrouj K, Colinge J, Gerbe F, Jay P, Krasinska L, et al. Cell-Cycle Regulation Accounts for Variability in Ki-67 Expression LevelsKi-67 Expression and the Cell Cycle. Cancer research. 2017;77(10):2722-34.
Decker T, Schneller F, Hipp S, Miething C, Jahn T, Duyster J, et al. Cell cycle progression of chronic lymphocytic leukemia cells is controlled by cyclin D2, cyclin D3, cyclin-dependent kinase (cdk) 4 and the cdk inhibitor p27. Leukemia. 2002;16(3):327-34.
Zhang M, Zhang L, Hei R, Li X, Cai H, Wu X, et al. CDK inhibitors in cancer therapy, an overview of recent development. American journal of cancer research. 2021;11(5):1913.
Knudsen ES, Kumarasamy V, Nambiar R, Pearson JD, Vail P, Rosenheck H, et al. CDK/cyclin dependencies define extreme cancer cell-cycle heterogeneity and collateral vulnerabilities. Cell reports. 2022;38(9):110448.
Li X-W, Rees JS, Xue P, Zhang H, Hamaia SW, Sanderson B, et al. New insights into the DT40 B cell receptor cluster using a proteomic proximity labeling assay. Journal of Biological Chemistry. 2014;289(21):14434-47.
Aleem E, Arceci RJ. Targeting cell cycle regulators in hematologic malignancies. Front Cell Dev Biol. 2015;3:16.
Solvason N, Wu WW, Parry D, Mahony D, Lam EW-F, Glassford J, et al. Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development. International immunology. 2000;12(5):631-8.
Chiles TC. Regulation and function of cyclin D2 in B lymphocyte subsets. The Journal of Immunology. 2004;173(5):2901-7.
Santamaria D, Barriere C, Cerqueira A, Hunt S, Tardy C, Newton K, et al. Cdk1 is sufficient to drive the mammalian cell cycle. Nature. 2007;448(7155):811-5.
Huertas P, Cortes-Ledesma F, Sartori AA, Aguilera A, Jackson SP. CDK targets Sae2 to control DNA-end resection and homologous recombination. Nature. 2008;455(7213):689-92.
Biggs JR, Peterson LF, Zhang Y, Kraft AS, Zhang DE. AML1/RUNX1 phosphorylation by cyclin-dependent kinases regulates the degradation of AML1/RUNX1 by the anaphase-promoting complex. Mol Cell Biol. 2006;26(20):7420-9.
Xu J, Wang YY, Dai YJ, Zhang W, Zhang WN, Xiong SM, et al. DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells. Proc Natl Acad Sci U S A. 2014;111(7):2620-5.
Richter A, Schoenwaelder N, Sender S, Junghanss C, Maletzki C. Cyclin-Dependent Kinase Inhibitors in Hematological Malignancies-Current Understanding, (Pre-)Clinical Application and Promising Approaches. Cancers. 2021;13(10).
Kubczak M, Szustka A, Blonski JZ, Gucky T, Misiewicz M, Krystof V, et al. Dose and drug changes in chronic lymphocytic leukemia cell response in vitro: A comparison of standard therapy regimens with two novel cyclin‑dependent kinase inhibitors. Mol Med Rep. 2019;19(5):3593-603.
Yue G. Inhibitory effect of indirubin on growth of some cancer cells and its mechanism. Tianjin Journal of Traditional Chinese Medicine. 2008.
Li Z, Wang H, Wei J, Han L, Guo Z. Indirubin exerts anticancer effects on human glioma cells by inducing apoptosis and autophagy. AMB Express. 2020;10(1):171.
Chen L, Wang J, Wu J, Zheng Q, Hu J. Indirubin suppresses ovarian cancer cell viabilities through the STAT3 signaling pathway. Drug Des Devel Ther. 2018;12:3335-42.
Perabo FG, Frossler C, Landwehrs G, Schmidt DH, von Rucker A, Wirger A, et al. Indirubin-3'-monoxime, a CDK inhibitor induces growth inhibition and apoptosis-independent up-regulation of survivin in transitional cell cancer. Anticancer research. 2006;26(3A):2129-35.
Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood. 2008;111(12):5446-56.
Zhang Y, Song L, Li J, Zhang Y, Lu X, Zhang B. Inhibitory effects of indirubin-3'-monoxime against human osteosarcoma. IUBMB Life. 2019;71(10):1465-74.
Kim SA, Kim YC, Kim SW, Lee SH, Min JJ, Ahn SG, et al. Antitumor activity of novel indirubin derivatives in rat tumor model. Clin Cancer Res. 2007;13(1):253-9.
Ribas J, Bettayeb K, Ferandin Y, Knockaert M, Garrofe-Ochoa X, Totzke F, et al. 7-Bromoindirubin-3'-oxime induces caspase-independent cell death. Oncogene. 2006;25(47):6304-18.
Lee MY, Liu YW, Chen MH, Wu JY, Ho HY, Wang QF, et al. Indirubin-3'-monoxime promotes autophagic and apoptotic death in JM1 human acute lymphoblastic leukemia cells and K562 human chronic myelogenous leukemia cells. Oncol Rep. 2013;29(5):2072-8.
Shi J, Shen HM. Critical role of Bid and Bax in indirubin-3'-monoxime-induced apoptosis in human cancer cells. Biochem Pharmacol. 2008;75(9):1729-42.
Shi J, Shen H-M. Indirubin-3’-monoxim induced apoptosis in human tumor cells via pro-apoptotic Bcl-2 family members. Clinical Cancer Research. 2014;13(19_Supplement):B46-B.
Hoessel R, Leclerc S, Endicott JA, Nobel ME, Lawrie A, Tunnah P, et al. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat Cell Biol. 1999;1(1):60-7.
Marko D, Schatzle S, Friedel A, Genzlinger A, Zankl H, Meijer L, et al. Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells. Br J Cancer. 2001;84(2):283-9.
Liao XM, Leung KN. Indirubin-3'-oxime induces mitochondrial dysfunction and triggers growth inhibition and cell cycle arrest in human neuroblastoma cells. Oncol Rep. 2013;29(1):371-9.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Indirubin in Vitro Apoptotic Effect Towards Chronic Lymphocytic Leukaemia (CLL) Lineage

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Subjects

2.2. Peripheral blood mononuclear cell (PBMC) isolation and cell culture

2.3. Cell viability assay

2.3. Apoptosis determination assay

2.4. RNA extraction, cDNA synthesis, and qRT-PCR

2.5. Statistical analysis

3. RESULTS

4. DISCUSSION

Declarations

References

Additional Declarations

Status:

Version 1