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ORIGINAL ARTICLE
Year : 2022  |  Volume : 18  |  Issue : 3  |  Page : 754-759

Enhanced expression of death receptor 5 is responsible for increased cytotoxicity of theophylline in combination with recombinant human TRAIL in MDA-MB-231 breast cancer cells


1 Department of Life Sciences, Presidency University, Kolkata, West Bengal, India
2 Department of Biotechnology, University of Engineering and Management, Kolkata, West Bengal, India

Date of Submission01-Mar-2021
Date of Acceptance31-Aug-2021
Date of Web Publication14-Jan-2022

Correspondence Address:
Ranjana Pal
Department of Life Sciences, Presidency University, 86/1 College Street, Kolkata - 700 073, West Bengal
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_352_21

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 > Abstract 


Background: Theophylline has been reported to induce cytotoxicity and cell cycle arrest in cancer cells. On the other hand, TRAIL, a secretory ligand, is known for its unique ability to induce cell death only in tumor cells. In the present study, we elucidated the mechanism behind the cytotoxic effect of theophylline in combination with recombinant human TRAIL (rhTRAIL) on cancer cell line MDA-MB-231.
Materials and Methods: Cytotoxicity of theophylline in combination with TRAIL was measured via trypan blue assay and MTT assay. Protein levels were assessed using Western hybridization. Reactive oxygen species (ROS) levels were measured using 2',7'–dichlorofluorescin diacetate and mitochondrial membrane potential (MMP) assay was conducted using tetramethylrhodamine, ethyl ester.
Results: We observed theophylline in combination with rhTRAIL to be significantly cytotoxic to the cancer cells in comparison to theophylline and rhTRAIL alone. Next, western hybridization showed combination treatment to upregulate cleaved form of caspase-8, 9 and 3, in comparison to the cells treated with rhTRAIL and theophylline alone. Theophylline in combination also increased the levels of ROS and reduced MMP. Interestingly, combination treatment increased the protein level of death receptor 5 (DR5), sensitizing the cells towards TRAIL-induced apoptosis.
Conclusion: Theophylline in combination with TRAIL significantly increases cytotoxicity in the MDA-MB-231 breast cancer cell line when compared to theophylline and rhTRAIL alone via upregulation of DR5 levels.

Keywords: Apoptosis, death receptor 5, recombinant human TRAIL, reactive oxygen species, theophylline


How to cite this article:
Tapadar P, Pal A, Dutta S, Pal R. Enhanced expression of death receptor 5 is responsible for increased cytotoxicity of theophylline in combination with recombinant human TRAIL in MDA-MB-231 breast cancer cells. J Can Res Ther 2022;18:754-9

How to cite this URL:
Tapadar P, Pal A, Dutta S, Pal R. Enhanced expression of death receptor 5 is responsible for increased cytotoxicity of theophylline in combination with recombinant human TRAIL in MDA-MB-231 breast cancer cells. J Can Res Ther [serial online] 2022 [cited 2022 Aug 10];18:754-9. Available from: https://www.cancerjournal.net/text.asp?2022/18/3/754/335488




 > Introduction Top


Theophylline even known as 1,3-dimethylxanthine, is a type of methylxanthine drug used for treating respiratory diseases for more than 80 years. It naturally occurs in tea and cocoa beans in trace amounts. In 1895, theophylline was first extracted from tea and chemically synthesized.[1] Theophylline is involved in some selective molecular mechanisms: Inhibition of mitosis, inhibition of phosphodiesterase, histone deacetylase-2 activity augmentation, reactive oxygen species (ROS) generation, and upregulation of interleukine-10 secretion.[2] Theophylline is also known to cause cytotoxicity and cell cycle arrest in breast cancer cells.[2],[3] In search of a more efficient way for the treatment of cancer, TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) has been studied extensively in the last two decades. Unlike other members of the TNF superfamily, the TNF-related apoptosis-inducing ligand (TRAIL, also known as Apo2L) possesses the unique capacity to induce apoptosis selectively in cancer cells, both in vitro and in vivo.[4],[5] Response of tumor cells to TRAIL is regulated by the presence of death receptors (DRs) or the decoy receptor (DCRs) on its cell surface. Upregulation of death receptor 5 (DR5) sensitizes the tumor cells toward TRAIL-induced apoptosis.[6] When TRAIL binds to DR5, this complex recruits FADD and caspase-8 for the formation of the death-inducing signaling complex.[7] Active caspase-8 can directly activate caspase-3 which induces apoptosis. Conversely, active caspase-8 can convert pro-caspase-9 into caspase-9, which in turn can activate caspase-3.[8] Mutation in the DR5 gene and/or endocytosis of DR5 protein from the plasma membrane are some of the reasons accountable for resistance against TRAIL-induced apoptosis in many cancer cells.[9],[10] TRAIL and DR5 agonistic antibodies have been used in clinical trials for different cancers.[11] Furthermore, it has been proven in preclinical trials that TRAIL is a secure and effective way of treating breast cancer patients.[12]

Till date, it is known that theophylline as well TRAIL can induce apoptosis in breast cancer cells. In the present study, we identified that theophylline in combination with recombinant human TRAIL (rhTRAIL) has a more pronounced effect on killing breast cancer cells compared to each of them alone. We further identified the mechanism behind the beneficial effect of theophylline in sensitizing breast cancer cells toward TRAIL-mediated apoptosis.


 > Materials And Methods Top


Recombinant human TRAIL protein induction and purification

TRAIL extracellular domain (aa 114–281) tagged with His-tag was expressed using pQE-hTR plasmid, a kind gift from Wafik El-Deiry (Addgene plasmid #21812).[13] The resulting 167aa active domain of TRAIL was regarded as rhTRAIL. pET-15b was used as a control plasmid. Induction and purification of rhTRAIL were performed as described by Kim et al.[13] Centrifugation of the resultant bacterial cultures was carried out at 6000 rpm for 30 min at 4°C followed by preparation of cell lysate using lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole, and 10 mM β-mercaptoethanol or 5 mM dithiothreitol.[14] Ni-NTA agarose beads was used for isolation of rhTRAIL from the soluble fraction after an initial wash with lysis buffer containing 20 mM imidazole. Isolated proteins were dialyzed against phosphate-buffered saline with 10 mM β-mercaptoethanol. Coomassie blue gel staining and immunoblotting were used to confirm the purity of the rhTRAIL protein.

Cell culture

MDA-MB-231, human breast adenocarcinoma cells were grown in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were maintained at humidified atmosphere 37°C with 5% CO2. Life Technologies supplied all the cell culture reagents and plastic wares, presently part of ThermoFisher Scientific, USA.

Cytotoxicity assay

The ability of rhTRAIL and theophylline to induce cytotoxicity at 24-h was determined using trypan blue dye exclusion assay.[2],[11],[15],[16] After 24-h of 10 mM theophylline (dissolved in 0.1M NaOH) treatment and 150 ng/ml rhTRAIL treatment, separately or in combination, cells were trypsinized and stained with trypan blue (Sigma-Aldrich, now part of Merck, Germany). Mock treatment was done using pET15b extract that had been purified and dialyzed along with 0.1M NaOH. This was used as control treatment for all the downstream experiments. Dead cells were counted via hemocytometer. Trypan blue assay has been done in quadruplicate and repeated thrice. The representative experiment is shown in [Figure 1]a.
Figure 1: Theophylline sensitizes breast cancer cells toward TRAIL-induced cell death. (a) Cytotoxicity was measured by trypan blue assay. (b) Cell viability was measured using MTT assay (C: Control; Tr: rhTRAIL; T: Theophylline; Tr + T: rhTRAIL + theophylline; *indicates P ≤ 0.05)

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Cell viability assay

Cell viability assay was done using thiazolyl blue tetrazolium bromide (MTT) reagent (SRL Pvt. Ltd., India). rhTRAIL and theophylline (Sisco Research Laboratories, India) were added separately or in combination to the wells at a final concentration of 150 ng/ml and 10 mM, respectively. Fresh media containing MTT reagent was added after 24-hrs of rhTRAIL and/or theophylline treatment.[2],[11],[15],[16] After 4 h of incubation, formazan crystals were dissolved in DMSO and the absorbance was measured at 590 nm on a SynergyH1 microplate reader with Gen5 software (Biotek, USA). MTT experiments have been carried out in triplicate and repeated thrice. The representative experiment is shown in [Figure 1]b.

Western blotting

Cells were treated with rhTRAIL and theophylline separately and in combination for 6-h. Next, cells were harvested and protein was isolated using RIPA buffer as per the manufacturer's instructions (Sigma-Aldrich). The whole protein extract was separated using SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (MerckMillipore, Germany). Proteins specific antibodies were used for detecting Caspase-9 (#9502), Caspase-3 (#9665), DR5 (#8074) and Caspase-8 (#D35G2) (Cell Signalling Technology, USA). β-actin (Biobharati LifeScience, India) was taken as endogenous control. To measure the intensity of each protein and to normalize using endogenous control, Image J software was used for each experiment. Graphs in [Figure 2] and [Figure 3] show Image J software analysis of average expression of four independent experiments normalized with respect to (w.r.t) β-actin.
Figure 2: Analysis of caspases on treatment of MDA-MB-231 cells with theophylline and/or rhTRAIL. (a) Level of cleaved caspase-8 (c.Casp8), (b) cleaved caspase-9 (c.Casp9) and (c) cleaved caspase-3 (c.Casp3) proteins as measured by immunoblot.(C: Control; Tr: rhTRAIL; T: Theophylline; Tr + T: rhTRAIL + Theophylline; *indicates P ≤ 0.05)

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Figure 3: Death receptor 5 expression in presence of theophylline and/or rhTRAIL. Expression levels of death receptor 5 protein was measured by immunoblot. β-actin was used as an endogenous control (C: Control; Tr: rhTRAIL; T: Theophylline; Tr + T: rhTRAIL + Theophylline; *indicates P ≤ 0.05)

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Reactive oxygen species 2',7'–dichlorofluorescin diacetate assay

As per the manufacturer's instructions, initially, cells were prestained with cell-permeant reagent 2',7'–dichlorofluorescin diacetate (Abcam, USA). Next, rhTRAIL and theophylline in previously mentioned concentration were added separately and in combination to the wells and incubated for 4-h. Fluorescent plate reader with excitation at 485 nm and emission at 535 nm was used for observation (SynergyH1, Biotek, USA). Experiments have been carried out in triplicate and repeated twice. The representative experiment is shown in [Figure 4]a.
Figure 4: Measurement of oxidative stress on the treatment of MDA-MB-231 cells with theophylline and/or rhTRAIL. (a) The level of reactive oxygen species was measured via DCFDA reagent. (b) Mitochondrial membrane potential was measured using tetramethylrhodamine, ethyl ester dye. (C: Control; Tr: rhTRAIL; T: Theophylline; Tr + T: rhTRAIL + Theophylline; *indicates P ≤ 0.05; RFU: Relative Fluorescent Units)

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Tetramethylrhodamine, ethyl ester-mitochondrial membrane potential assay

After 6-h of treatment, active mitochondria were labeled with tetramethylrhodamine, ethyl ester. The manufacturer's instruction was followed to carryout the rest of the experiment (Cell Signalling Technology, USA). Next, the prepared samples were analyzed on a plate reader at excitation of 550 nm and emission of 580 nm (SynergyH1, Biotek, USA). Experiments have been carried out in triplicate and repeated twice. The representative experiment is shown in [Figure 4]b.

Data analysis

All the shown comparisons between different groups were performed via the unpaired two-tailed t-test using SPSS statistical software (SPSS Inc., Chicago, IL, USA). Significant P value was considered at and <0.05.


 > Results Top


Combined treatment with theophylline and recombinant human TRAIL enhanced cytotoxicity and reduced cell viability in MDA-MD-231 cells compared to theophylline or recombinant human TRAIL alone

To investigate the cytotoxic effect of theophylline in combination with rhTRAIL, cell death, and cell viability assay was performed in Triple-Negative Breast Cancer (TNBC) cell line MDA-MB-231. Cells were treated with rhTRAIL and theophylline separately and in combination for 24 h. rhTRAIL alone showed 24.4% of cell death and theophylline itself induced 36.4% cell death. In combination of rhTRAIL and theophylline, 51.3% cell death was induced [Figure 1]a. In cell viability assay, rhTRAIL and theophylline separately showed 70.6% and 54.6% cell viability, in combination the cell viability reduced to 40.5% [Figure 1]b.

Combination treatment of theophylline and recombinant human TRAIL induced both extrinsic and intrinsic pathways of apoptosis better than theophylline or recombinant human TRAIL alone

We further observed that theophylline in combination with rhTRAIL significantly increased cleaved caspase-8 protein level after 6 h of treatment in comparison to theophylline or rhTRAIL alone [Figure 2]a. As TRAIL is known for inducing both the branches of apoptosis, we studied the intrinsic apoptotic genes such as cleaved caspase-9 and the final effector protein of apoptosis pathway, caspase-3. Both of the cleaved caspases are significantly upregulated in cells treated with theophylline and rhTRAIL alone as well as in combination compared to control cells. Furthermore, the cleaved caspase-9 and caspase-3 in combination are higher than those of theophylline and rhTRAIL treated sample [Figure 2]b and [Figure 2]c.

Theophylline in combination with recombinant human TRAIL induced high reactive oxygen species production along with enhanced lipid peroxidation in comparison to theophylline or recombinant human TRAIL alone

To gain insight into the mechanism of cytotoxicity mediated via theophylline in combination with rhTRAIL, we observed the level of ROS production in MDA-MB-231 cells treated with theophylline and/or rhTRAIL. ROS generation was found to be significantly upregulated in cells treated with the combination of rhTRAIL and theophylline, in comparison to rhTRAIL and theophylline separately [Figure 4]a. In addition, we observed mitochondrial membrane potential (MMP) to be lower in combination treatment compared to single treatment of rhTRAIL and theophylline [Figure 4]b.

Theophylline upregulated death receptor 5 expression in combination with recombinant human TRAIL with respect to the single treatment

To understand the mechanism by which apoptosis is induced by combination treatment, we performed western blot analysis for DR5 protein. We found that expression of DR5 was increased significantly on the addition of theophylline with rhTRAIL with respect to MDA-MB-231 cells that were exposed to theophylline or rhTRAIL alone [Figure 3].


 > Discussion Top


Theophylline as reported to be toxic for cancer cells opens a new pathway for alternative drug combination therapy. With promising anti-cancer properties, TRAIL is regarded as a potential therapeutic agent. TRAIL sensitivity differs among various cancer types. The most malignant tumors being resistant to TRAIL treatment gives more reason for finding new molecules to sensitize these cells towards TRAIL-induced apoptosis.[17] In our study, we found that theophylline in combination with rhTRAIL induces significant cell death in comparison to rhTRAIL and theophylline alone [Figure 1]a and [Figure 1]b. The extrinsic apoptosis cascade was activated via upregulation of cleaved caspase-8 in all the treated cells compared to control cells [Figure 2]a. We also confirm the activation of the intrinsic pathway, via western blot analysis for caspase-9 and the final molecule of apoptotic pathway caspase-3. Both the cleaved form of the caspases were significantly upregulated in cells treated with the combination of theophylline and rhTRAIL compared to single treatment with either rhTRAIL or theophylline [Figure 2]b and [Figure 2]c.

ROS generation disrupts the MMP, causing activation of the intrinsic apoptotic pathway.[18] We investigated the ROS production level in cells on treatment with rhTRAIL and/or theophylline where we observed that the amount of ROS is significantly higher in combination with respect to rhTRAIL and theophylline alone [Figure 4]a. In continuation of the pathway, we also found MMP levels are lower in cells treated with the combination dose [Figure 4]b.

Studies have shown upregulation of DR5 sensitizes tumor cells toward TRAIL-mediated apoptosis.[19] Interestingly, we found that DR5 expression level was increased in combination samples in comparison to rhTRAIL and theophylline alone [Figure 3]. Reports have shown that ROS upregulates DR5 expression in various cancer types and DR5 can induce apoptosis via both extrinsic and intrinsic apoptotic pathway.[20],[21],[22] ROS is known to activate biological processes or initiate oxidative stress depending on its concentration in a cellular system.[23] Here, we hypothesize that combination treatment increases ROS at a threshold that invokes upregulation of DR5 protein. This increase in abundance of TRAIL receptor, DR5, in combination treatment increases the chance of rhTRAIL binding to its cognate receptor thereby intensifying apoptosis [Figure 5].
Figure 5: Graphical representation of apoptosis pathway induced on exposure of cancer cells to rhTRAIL and/or theophylline. rhTRAIL on binding to death receptor 5 activates apoptosis via caspase-8, -9 and -3. Theophylline increases reactive oxygen species and caspases thereby inducing apoptosis. Combination treatment causes overexpression of the death receptor 5. We hypothesize that in combination treatment reactive oxygen species, produced on theophylline exposure, crosses a threshold which in turn induces death receptor 5 expression thereby activating caspase-mediated apoptosis

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 > Conclusion Top


We can say that the combination of theophylline and rhTRAIL causes significant cytotoxicity via upregulation of DR5 which in turn induces enhanced apoptosis in cancer cells compared to individual treatment. Our study indicates that theophylline in combination with TRAIL can be further explored for treating TNBC patients.

Acknowledgment

I would like to acknowledge Dr. Sugopa Sengupta and Dr. Nabendu Biswas of Presidency University, Kolkata, India for providing pET-15b control plasmid and caspase-8 antibody, respectively. The MDA-MB-231 cell line was a kind gift from Prof. Gaurisankar Sa of Bose Institute, Kolkata, India.

Financial support and sponsorship

This research was supported by grant from West Bengal Department of Science and Technology (WB-DST; Memo No. 152(Sanc.)/ST/P/S and T/9G-22/2017) to R.P. Infrastructure support was made possible with funds from the Presidency University Funds, Department of Biotechnology- Boost to University Interdisciplinary Life Science Departments for Education and Research Programme (DBT-BUILDER) and Department of Science and Technology - Fund for Improvement of S and T Infrastructure in Higher Educational Institutions (DST-FIST). P.T. and A.P. were supported by DST-INSPIRE fellowship and WB-DST, respectively.

Conflicts of interest

There are no conflicts of interest.



 
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Kim SH, Kim K, Kwagh JG, Dicker DT, Herlyn M, Rustgi AK, et al. Death induction by recombinant native TRAIL and its prevention by a caspase 9 inhibitor in primary human esophageal epithelial cells. J Biol Chem 2004;279:40044-52.  Back to cited text no. 13
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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