Therapeutic natural compounds Enzastaurin and Palbociclib inhibits MASTL kinase activity preventing breast cancer cell proliferation

doi:10.21203/rs.3.rs-1254395/v1

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Research Article

Therapeutic natural compounds Enzastaurin and Palbociclib inhibits MASTL kinase activity preventing breast cancer cell proliferation

https://doi.org/10.21203/rs.3.rs-1254395/v1

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Microtubule-associated serine/threonine kinase-like (MASTL) regulates mitotic progression and is an attractive target for the development of new anticancer drugs. In this study, novel inhibitory molecules were screened against MASTL kinase, a protein involved in cell proliferation in breast cancer. Natural source-derived drugs Enzastaurin and Palbociclib were selected to identify their role as MASTL kinase inhibitors. Cytotoxic activity, kinase activity and other cell-based assays of Enzastaurin and Palbociclib were evaluated on human breast cancer (MCF-7) cells. The potential natural compounds caused cytotoxicity in MCF-7 cells in a dose and time-dependent manner. Further analysis by Annexin V and PI staining indicated that both drugs are potent inducers of apoptosis. Enzastaurin induced G2/M phase arrest while, Palbociclib caused G1 arrest. MASTL kinase activity was significantly abrogated with both the compounds showing EC₅₀ values of 17.13µM and 10.51µM respectively. Taken together, these data strongly suggest that Enzastaurin and Palbociclib possess the ability to inhibit MASTL kinase activity and induce cell death in breast cancer cells, thus exhibiting significant therapeutic potential.

Breast cancer

Microtubule-associated serine/threonine kinase (MASTL)

drug discovery

therapeutics

in-vitro kinase assay

The Serine/Threonine Protein Kinase subfamily includes AGC kinases and Microtubule-associated serine/threonine kinase (MASTL) or Great wall kinase (GWL) is a member of the AGC family of kinases. The huge number of proteins that AGC kinases can phosphorylate demonstrates their importance in a variety of cellular activities. Serine /threonine kinases are essential regulators of cell adhesion and contraction, which is important for cancer growth and metastasis [1]. Many human disorders, including cancer, are caused by mutations or dysregulation of AGC kinases, which mediates a wide range of critical cellular processes [2].

MASTL is a unique AGC kinase that lacks a hydrophobic motif, unlike most AGC kinases, despite the presence of a hydrophobic pocket that specifies its particular method of regulation [3]. It features a unique T-loop region with insertion of roughly 500 amino acids. It has received far less attention as compared to other AGC kinases. Furthermore, in immortalized normal breast epithelial cells, overexpression of MASTL slows cell cycle progression, causes abnormal cell division, DNA damage response, affects migration, the actin cytoskeleton, and cell–cell junctions, tumor resistance in response to anticancer treatments and leads to enhanced invasion and metastasis in vitro and in vivo, leading to cancer development [4]. Moreover, MASTL triggered mitotic cell death in a variety of cancer cells while normal cells were less affected [5]. It is known to be a key player in the cell division, growth, metabolism, and differentiation. It accelerates the cell cycle progression by phosphorylating Endosulfine Alpha (ENSA) and Arpp19, which limits PP2A-B55 phosphatase activity and hence maintains the phosphorylated status of Cyclin-dependent kinase 1 (CDK1) substrates [6]. MASTL is phosphorylated during mitosis which is an essential requirement for its activation [7,8]. MASTL inhibition of PP2A-B55 is essential to sustain the mitotic state during cell division, whereas MASTL inactivation and PP2A reactivation is required for mitotic departure [9]. In vitro and in vivo studies in breast cancer found that inhibiting MASTL reduced tumour development and metastasis. These findings suggest that MASTL is a new breast cancer oncogene that may overcome contact inhibition, invasion, and chromosomal instability [10]. Overall, the evidence revealed that MASTL can be a promising target for selective anticancer treatment [11].

Research have focused on the role of natural origin drugs in anticancer treatment due to the limited number of adverse effects and the wide range of targets of naturally-derived components [12]. Till date, a few studies have reported MASTL inhibitors like GK-1, MKI-1, MKI-2 and a thieno-pyrimidinone based tricyclic derivative. These have been identified and validated using virtual screening and in-vitro analysis GKI-1 inhibited MASTL in vitro and interrupted mitotic events by lowering phosphorylated ENSA with µM range potency in HeLa cells. Moreover, in other breast cancer cells, GKI-1 exhibited negligible anti-cancer effect [13]. In in vitro and in vivo models of breast cancer, MKI-1 was found to have anticancer and radiosensitizer properties. MKI-1 demonstrated µM range potency and efficacy for MASTL inhibition. In addition, MKI-1 reduced the amount of c-Myc protein in breast cancer cells through increasing PP2A activity [14]. Recently, it was found that MKI-2 decreased recombinant MASTL activity and induced mitotic catastrophe in breast cancer cells through modulating the MASTL-PP2A axis. In mouse oocytes that were employed as a model to validate MKI-2 activity, the MKI-2 treatment displayed phenocopies with MASTL-null oocytes. MKI-2 inhibited MASTL in breast cancer cells with potency and effectiveness in the nM range [15]. All these identified MASTL inhibitors are synthetic and their toxicity profiles and long-term effects are yet to be studied. Our group has also previously identified various compounds from both natural and synthetic sources, ZINC85597499 and ZINC53845290 using virtual screening that proved to be significant leads for further experimental validation [16].

The present study is focused on the identification of new MASTL inhibitors from natural sources. We have identified Enzastaurin and Palbociclib as MASTL kinase inhibitors using in-vitro kinase assay. Moreover, these compounds also demonstrated an anti-proliferative effect on breast cancer cells. Both the compounds exhibited potency and inhibition efficacy in the µM range (1.56µM to 50µM). In addition, we investigated the influence on cell-cycle progression and apoptosis as an anti-proliferative effect was observed in MCF-7 cells treated with these natural products. Cell cycle study revealed that the Enzastaurin and Palbociclib are capable of arresting the cells in G2/M and G1 phases respectively. Thus, in the present study we have identified naturally derived compounds Enzastaurin and Palbociclib as novel MASTL kinase inhibitors with significant antitumor effect as potential therapeutic leads against breast cancer that can further be validated in animal models.

Cell culture

MCF-7 cell line purchased from NCCS, Pune was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. The cell line was maintained in water-jacketed CO₂ incubator (Forma series 2, Thermofisher, India) at 37ºC and 5% CO₂. To ensure culture was free from mycoplasma contamination, cells were tested and authenticated. The cells were sub-cultured routinely, every three to four days [17].

Luminescence-based in-vitro kinase assay

MASTL kinase enzyme system kit manufactured by Promega Corporation was used to perform this assay and protocol was followed as per the manufacturer’s specifications. The MASTL kinase inhibitor, Enzastaurin (1.56µM to 50µM) and Palbociclib (1.56µM to 50µM) along with MASTL kinase (25ng), substrate (0.1µg/µl) and ATP (10µM) were diluted separately in 1X kinase buffer. All the solutions were mixed in a 96 well-plate, solid white, round bottom, low volume (Corning, India). After incubating the mixture at room temperature for 1 hr, 25µl of ADP-Glo Reagent was added and incubated at room temperature for 40 mins to terminate the kinase reaction. This was followed by adding 50µl of Kinase detection reagent to convert ADP to ATP. The solution was further, incubated at room temperature for 30 mins. After incubation, the newly synthesized ATP was measured as luminescence value in a multimode microplate reader (Spark, Tecan Lifesciences) [18].

Cell viability assay

MCF-7 (1 x 10⁴) cells at 70-80% confluency was plated in triplicates in 96-well plate (Costar, Corning, India). Cells were allowed to adhere for 24 hrs in 100µl of DMEM per well. Cells were treated with Enzastaurin and Palbociclib in a dose-dependent manner (1.56µM to 25µM) for 24 hrs in CO₂ incubator. Cells without any drug treatment was used as control. After 24 hrs of incubation, complete media was removed and cells were washed with 1X PBS. 20µl MTT (5 mg/ml) was added and further, incubated for 2hrs [19]. MTT was aspirated and formazan produced was dissolved in 100µl of 0.1% DMSO. OD (optical density) values were taken at 570 nm (630 nm as a reference wavelength) by using DMSO as blank.

Annexin V/Propidium iodide apoptosis assay

MCF-7 (2x10⁵) cells were seeded per well in a 6-well plate in 2ml media (in triplicates) overnight. Cells were treated with Enzastaurin (6.25µM to 25µM) and Palbociclib (3.12µM to 12.5µM) in a dose-dependent manner for 24 hrs. The assay was performed using the Annexin V/Propidium iodide apoptosis assay kit (V13241, Invitrogen) according to the manufacturer’s protocol [20]. Flow cytometry was performed on a BD FACS Lyric flow cytometer (BD Biosciences, India). Data was generated for 10,000 events per sample and analysed using BD FACS Suite software (BD Biosciences, India) [21].

Cell cycle assay

MCF-7 2x10⁵/2ml cells plated in 6-well plate were treated with Enzastaurin (6.25µM to 25µM) and Palbociclib (3.12µM to 12.5µM) for 24 hrs. Cells were washed in chilled1X PBS after trypsinization. Cells were fixed in 70% ethanol for 30 mins at 4ºC. The centrifuged cell pellet after incubation was suspended in 1X PBS. Further, cell pellet was resuspended in 50 µg/ml of propidium iodide and 10µg/ml of RNAse A prepared in 1X PBS. The cells were incubated in dark for 30 mins before data acquisition [22]. Further, samples were examined on a BD FACS Lyric flow cytometer (BD Biosciences, India). Data analysis was performed using BD FACS Suite software (BD Biosciences, India) with 10,000 events per sample.

MASTL kinase assay of novel inhibitors

The effects of natural drugs on MASTL activity were investigated by in vitro kinase assay using ADP-Glo luminescent assay kit. A significant decrease was observed in the luminescence values of MASTL kinase activity with an increase in the concentration of drugs as shown in Fig. 1a.

The luminescent signal generated was correlated with the kinase activity. Incubation of MASTL kinase with 50µM concentration of Enzastaurin and Palbociclib, resulted in 66% and 71% of inhibition. This clearly reflects that MASTL kinase activity decreased with an increase in the concentration of both the compounds as compared to control.

The EC₅₀ (effective concentration) value for inhibitors, was calculated from the percent (%) kinase activity values using the Graphpad Prism software. Thus, the luminescence-based in-vitro kinase assay established EC₅₀ for Enzastaurin and Palbociclib as 17.13µM and 10.51µM respectively (Fig. 1b). This signifies that Palbociclib exhibits a better inhibitory kinase activity against MASTL.

Enzastaurin and Palbociclib inhibited proliferation of MCF-7 cells

The cytotoxic effect of Enzastaurin and Palbociclib inhibitors from natural sources on MCF-7 cells was monitored by MTT assay. Cells treated with compounds for 48 hrs in a dose-dependent manner showed a decrease in the percentage of cell viability (Fig. 2a). The cell viability decreased from 94.4% at 1.56µM to 44.2% at 25µM of Enzastaurin as compared to control (without drug treatment). On the other hand, the percentage of cell viability in Palbociclib treated cells decreased from 93.7% at 1.56µM to 4.4% at 25µM. The IC₅₀ value of Enzastaurin and Palbociclib was 19.18µM and 5.22µM, respectively (Fig. 2b). IC₅₀ dose and sublethal doses for both the compounds were selected for further experimentation.

Enzastaurin and Palbociclib induced apoptotic cell death in MCF-7 cells

The effect of Serine/Threonine kinase inhibitors on apoptosis induction in MCF-7 cells was examined. MCF-7 cells were exposed to various concentrations of the compounds for 24 hrs and apoptosis was detected. Flow cytometric analysis with Annexin-V and PI fluorescent staining was used to quantify the induction of apoptosis by natural inhibitors.

The number of apoptotic cells increased in a dose-dependent manner from 13.6% at 6.25µM to 45.5% at 25µM upon Enzastaurin treatment as compared to control cells. A similar increase in apoptotic cells was observed with Palbociclib but the effect was less prominent in comparison to Enzastaurin, whereby, 23.3% apoptosis was seen with 12.5µM Palbociclib as depicted in Fig. 3. This signifies that the natural compounds are capable of inducing apoptosis in MCF-7 cells in a dose-dependent manner.

Newly identified natural compounds induce cell cycle arrest in MCF-7 cells

To evaluate the effect of natural compounds on cellular proliferation, the cell cycle distribution of MCF-7 cells was assessed. MCF-7 cells treated with Enzastaurin exhibited a significant increase in G2/M phase population as revealed by cell cycle analysis, while Palbociclib arrested cells in G1 phase (Fig. 4a & 4b). Approximately, 24% of cells were arrested in G2/M at 25 µM Enzastaurin and 55% in G1 with 12.5µM Palbociclib.

Interestingly, Palbociclib exhibits a better inhibitory activity than Enzastaurin. The incubation of MCF-7 cells in the presence of Enzastaurin substantially arrested the cell cycle in the G2/M phase and Palbociclib treatment significantly arrested the cells in the G1 phase (as shown in Fig. 4a & 4b) by inhibiting the cell cycle progression at the initial checkpoint itself.

Studies investigated MASTL to be an important drug target for anticancer treatment due to its multifarious roles such as cellular transformation, metastasis, chromosomal instability, and the DNA damage response in various cancers [10,11, 24]. MASTL is reported to be upregulated in several cancer cell lines including breast cancer cells [4, 25-27]. Furthermore, MASTL upregulation is also strongly correlated with poor survival in breast cancer patients [28]. MASTL depletion induced cell death in MCF7 breast cancer cells [29]. These studies suggest the potential role of MASTL in cancer cells survival and proliferation. Hence, identifying the new natural inhibitors against MASTL will be an attractive strategy for cancer therapy. In-silico screening and in-vitro analysis have previously revealed GK-1, MKI-1, and MKI-2 as potential MASTL inhibitors. GK-1 and MKI-1 showed potency in µM range while the inhibitory potential of MKI-2 was in nM range [13-15]. Previously, our group has also computationally identified and validated different inhibitors, ZINC85597499 and ZINC53845290 against this protein from both the natural and synthetic origin [16]. Till date, no natural MASTL inhibitor with antitumor activity has been discovered. Thus, in the current research, compounds of natural origin were chosen to target MASTL protein in the cancerous cells. Enzastaurin and Palbociclib were explored as MASTL inhibitors with anticancer activity against breast cancer cells. Enzastaurin and Palbociclib showed MASTL kinase inhibition in a dose-dependent manner with EC₅₀ value of 17.13µM and 10.51µM respectively. Further, we investigated the cytotoxic activities of these natural products on MCF-7 cell proliferation and found the IC₅₀ values of Enzastaurin and Palbociclib to be 19.18µM and 5.22µM.

The compounds caused MCF-7 cells to arrest in different phases of cell cycle. Palbociclib, a mimic of natural product [30] remarkably, arrested cells in the G1 phase and on the other hand, Enzastaurin, an analog of Staurosporine isolated from Streptomyces sp. [31] showed G2/M phase arrest. Interestingly, Palbociclib significantly slowed cell cycle advancement in the G1 phase by suppressing the progression at the initial checkpoint. Further, our data showed that drugs were effective in inducing apoptosis in a dose-dependent way with respect to the control cells showing 45.5% and 23.3% of apoptotic cells at the highest dosage of Enzastaurin (25 µM) and Palbociclib (12.5 µM).

Therefore, our data strongly indicate Enzastaurin and Palbociclib as new natural inhibitors of MASTL kinase. In addition, we also observed anticancer activity with both the compounds as they inhibited cell proliferation and induced apoptosis in breast cancer cells. Further animal-based studies with these compounds will provide mechanistic insights and establish these compounds as promising leads for breast cancer treatment.

MASTL- Microtubule assisted serine/threonine kinase, GWL- Great wall kinase, ENSA - Endosulfine Alpha, CDK1- Cyclin-dependent kinase 1, PI- Propidium iodide, IC₅₀- Inhibitory concentration, EC₅₀– Effective concentration

Acknowledgments

Dr. Anupkumar R. Anvikar, Director, NIB, Noida is deeply acknowledged for his continuous support and guidance. AP acknowledges the infrastructural support from National Institute of Biologicals (NIB), Noida. JR acknowledges the Department of Science & Technology (DST) for awarding the DST-WOS-A fellowship (No. SR/WOS-A/LS-161/2018 (G)) and thanks Prof. Monisha Banerjee for infrastructural support. We would like to thank Dr. Anil Mishra, Director, Institute of Nuclear Medicine & Allied Sciences (INMAS), Delhi for the continuous infrastructural support. We would like to thank Dr. Sankar Bhattacharyya, Group Leader in Vaccine Development, Premas Biotech Pvt. Ltd., Gurugram, Haryana for his critical review of the manuscript.

Conflicts of Interest

There are no potential conflicts of interest declared by the authors.

Funding

GM acknowledges the grant from the Indian Council of Medical Research (ICMR) for awarding an Extramural Ad-hoc research project (Project ID: 2019_3182).

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

Gauri Misra: Conceptualization, Methodology, Manuscript Compilation; Aneesha Polisety: Data curation, Methodolgy, Manuscript compilation; Jyotika Rajawat: Methodology, Manuscript Compilation; Amit Katiyar: Software, Data curation; Harpreet Singh: Data analysis; Anant Narayan Bhatt: Data Analysis

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.

Ethics approval: The manuscript does not contain clinical studies or patient data.

Consent to participate: Not applicable

Consent to publish: Not applicable

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Therapeutic natural compounds Enzastaurin and Palbociclib inhibits MASTL kinase activity preventing breast cancer cell proliferation

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Abstract

Figures

Introduction

Methods

Results

MASTL kinase assay of novel inhibitors

Enzastaurin and Palbociclib inhibited proliferation of MCF-7 cells

Enzastaurin and Palbociclib induced apoptotic cell death in MCF-7 cells

Newly identified natural compounds induce cell cycle arrest in MCF-7 cells

Discussion

Abbreviations

Declarations

References

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