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Curcumin augments therapeutic efficacy of TRAIL-based immunotoxins in leukemia

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Abstract

Background

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been perceived as a promising anti-cancer agent because of its unique ability to kill cancer cells while sparing normal cells. However, translation of TRAIL to clinical studies was less successful as a large number of cancer cells acquire resistance to TRAIL-based monotherapies. An ideal strategy to overcome TRAIL resistance is to combine it with potential sensitizing agents.

Objective

To investigate the TRAIL-sensitizing effect of curcumin in leukemia.

Methods

The mechanism underlying TRAIL sensitization by curcumin was studied by flow cytometric analysis of TRAIL receptors in leukemic cell lines and patient samples, and immunoblot detection of TRAIL-apoptosis signaling proteins.

Results

Curcumin augments TRAIL-apoptotic signaling in leukemic cells by upregulating the expression of DR4 and DR5 along with suppression of cFLIP and anti-apoptotic proteins Mcl-1, Bcl-xl, and XIAP. Curcumin pre-treatment significantly (p < 0.01) enhanced the sensitivity of leukemic cell lines to TRAIL recombinant proteins. IL2-TRAIL peptide in the presence of curcumin induced potent apoptosis (p < 0.001) as compared to TRAIL and IL2-TRAIL protein in leukemic cell lines with IC50 < 0.1 μΜ. Additionally, the combination of IL2-TRAIL peptide and curcumin showed significant cytotoxicity in patient peripheral blood mononuclear cells (PBMCs) with an efficacy of 90% in acute myeloid leukemia (AML), but 100% in acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and chronic myelomonocytic leukemia (CMML).

Conclusion

Overall, our results suggest that curcumin potentiates TRAIL-induced apoptosis through modulation of death receptors and anti-apoptotic proteins which significantly enhances the therapeutic efficacy.

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Acknowledgements

The authors wish to thank Apollo Cancer Specialty Hospital, Chennai for providing the human peripheral blood samples and for extending their diagnostic flow cytometry facility for sample analysis.

Funding

This work was supported by the Department of Science and Technology (DST), Government of India (No: SR/SO/HS-016/2013).

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Author notes

  1. Madhumathi Jayaprakasam

    Present address: Division of Epidemiology and Communicable Diseases, Indian Council for Medical Research (ICMR), New Delhi, India

Authors and Affiliations

  1. Stem Cell and Molecular Biology Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences Building, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India

    Sridevi Surapally & Rama Shanker Verma

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Correspondence to Rama Shanker Verma.

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The authors declare that they have no conflict of interest in the present study.

Ethical approval

Prior approval was obtained from the Institutional Ethics Committee, IIT Madras (IEC-IITM) for the study including patient blood samples. The ethics committee approval reference number for the present study is IEC/2-16/01/RSV-5/21. All experiments involving human participants have been performed in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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ESM 1 Surface analysis of decoy receptors in curcumin treated leukemic cell lines.

Flow cytometry analysis to quantify DcR1 and DcR2 receptors expression in leukemic cell lines was performed after 24 h curcumin treatment. Cells (5 ×105) were exposed to 25 μM of curcumin for 24 h, stained with 1 μg/ml anti-DcR1-PE or anti-DcR2 followed by 1hr incubation with goat anti-mouse Alexa Fluor® 594 secondary and analyzed by flow cytometry. Panel (a) represents the DcR1 expression while panel (b) represents the DcR2 expression of leukemic cell lines. Cells without any staining were used as a negative control. Isotype controls of the respective treated conditions were used for gating analysis. 1 (TIF 11030 kb)

ESM 2 Statistical analysis of Annexin V-FITC/PI staining.

Apoptosis induced by TRAIL, IL2-TRAIL and IL2-TRAIL peptide alone and in combination with curcumin in K562 cell line was assessed by dual staining with Annexin V-FITC/PI staining.  Annexin-V-positive or Annexin-V/PI-double-positive cells were considered apoptotic. The data is presented as mean values from three separate experiments ± SEM. The statistical analysis was done using Graphpad prism software version 5. Two-way ANOVA was used with Bonferroni’s post-test. A probability (p) value of < 0.05 was considered statistically significant. 2 (TIF 796 kb)

ESM 3 Comparison of surface receptor expression in patient samples.

Comparison of mean expression levels of DR4 and DR5 receptors in curcumin treated and untreated conditions in different leukemic samples a) ALL, b) AML, c) CLL and d) CMML. Surface levels of receptors DR4 and DR5 in different leukemic samples were assessed by flow cytometry. Each data point represents expression in one patient sample in terms of percentage of cells. Mean ± SEM of different samples is represented. 3 (TIF 301 kb)

ESM 4 Real-time PCR analysis of transcript levels of DR4 and DR5 receptors in curcumin treated patient samples.

Gene expression analysis of DR4 and DR5 receptors in curcumin (25 μM) treated patient samples (a) acute lymphoblastic leukemia (ALL) (b) acute myeloid leukemia (AML), (c) chronic lymphocytic leukemia (CLL) and (d) chronic myelomonocytic leukemia (CMML) samples. RNA from untreated peripheral blood mononuclear cells (PBMCs) was used for normalization of the respective samples. In a, b and c data represents mean ± SEM of transcription levels of three different experiments while mean values alone were represented in CMML. Each data point represents expression in one patient sample. 4 (TIF 2330 kb)

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Surapally, S., Jayaprakasam, M. & Verma, R.S. Curcumin augments therapeutic efficacy of TRAIL-based immunotoxins in leukemia. Pharmacol. Rep 72, 1032–1046 (2020). https://doi.org/10.1007/s43440-020-00073-7

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