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TriCurin, a synergistic formulation of curcumin, resveratrol, and epicatechin gallate, repolarizes tumor-associated macrophages and triggers an immune response to cause suppression of HPV+ tumors

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Abstract

Our earlier studies reported a unique potentiated combination (TriCurin) of curcumin (C) with two other polyphenols. The TriCurin-associated C displays an IC50 in the low micromolar range for cultured HPV+ TC-1 cells. In contrast, because of rapid degradation in vivo, the TriCurin-associated C reaches only low nano-molar concentrations in the plasma, which are sub-lethal to tumor cells. Yet, injected TriCurin causes a dramatic suppression of tumors in TC-1 cell-implanted mice (TC-1 mice) and xenografts of Head and Neck Squamous Cell Carcinoma (HNSCC) cells in nude/nude mice. Here, we use the TC-1 mice to test our hypothesis that a major part of the anti-tumor activity of TriCurin is evoked by innate and adaptive immune responses. TriCurin injection repolarized arginase1high (ARG1high), IL10high, inducible nitric oxide synthaselow (iNOSlow), IL12low M2-type tumor-associated macrophages (TAM) into ARG1low, IL10low, iNOShigh, and IL12high M1-type TAM in HPV+ tumors. The M1 TAM displayed sharply suppressed STAT3 and induced STAT1 and NF-kB(p65). STAT1 and NF-kB(p65) function synergistically to induce iNOS and IL12 transcription. Neutralizing IL12 signaling with an IL12 antibody abrogated TriCurin-induced intra-tumor entry of activated natural killer (NK) cells and Cytotoxic T lymphocytes (CTL), thereby confirming that IL12 triggers recruitment of NK cells and CTL. These activated NK cells and CTL join the M1 TAM to elicit apoptosis of the E6+ tumor cells. Corroboratively, neutralizing IL12 signaling partially reversed this TriCurin-mediated apoptosis. Thus, injected TriCurin elicits an M2→M1 switch in TAM, accompanied by IL12-dependent intra-tumor recruitment of NK cells and CTL and elimination of cancer cells.

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Abbreviations

ARG1:

Arginase-1

C:

Curcumin

CD8:

Cluster of differentiation 8 protein

CD8α:

Cluster of differentiation 8 alpha

CTL:

Cytotoxic T lymphocytes

CUNY:

City University of New York

E:

Epicatechin gallate

E6 16/18:

Early 6 protein 16/18

E7:

Early 7 protein

HaRas:

Harvey ras protein

HCl:

Hydrochloric acid

HNSCC:

Head and neck squamous cell carcinoma

HPV:

Human papillomavirus

IACUC:

Institutional Animal Care and Use Committee

Iba1:

Ionized calcium-binding adapter molecule 1

IF:

Integrated fluorescence

IFNγ:

Interferon gamma

IgG:

Immunoglobin G

IL10:

Interleukin-10

IL12:

Interleukin-12

iNOS:

Inducible nitric oxide synthase

LEEP:

Loop electrosurgical excision procedure

LR:

Lower right

NCr:

Athymic nude mice

NKp46:

Natural killer cell p-46-related protein

NOS2:

Nitric oxide synthase 2

PFA:

Paraformaldehyde

P-NF-kB:

Phospho-NF-kB

P-p65:

Phospho-p65

P-ser:

Phosphor-serine

P-STAT1/3:

Phospho-STAT1/3

P-Tyr:

Phospho-tyrosine

R:

Resveratrol

S.E.:

Standard error

SEM:

Standard error of the mean

SSC:

Saline-sodium citrate

TAM:

Tumor-associated macrophage

TriCur + IL12Ab:

‘IL12 antibody infusion followed by TriCurin treatment’ group

UL:

Upper left

UMSCC47:

University of Michigan-squamous cell carcinoma-47

UR:

Upper right

w/v:

Weight/volume

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Acknowledgements

Sumit Mukherjee was supported by a teaching assistantship from the College of Staten Island (CUNY).

Funding

This project was supported by Professional Staff Congress (PSC)-CUNY Grants from cycles 45 and 47.

Author information

Author notes

  1. Rahman Hussaini and Richard White contributed equally.

Authors and Affiliations

  1. CUNY Doctoral Program in Biochemistry, CUNY Graduate Center, New York, NY, 10016, USA

    Sumit Mukherjee & Angela Fried

  2. Department of Chemistry, Building 6S, The City University of New York at The College of Staten Island, 2800 Victory Boulevard, Staten Island, NY, 10314, USA

    Sumit Mukherjee & Probal Banerjee

  3. The Center for Developmental Neuroscience, City University of New York at The College of Staten Island, 2800 Victory Boulevard, Staten Island, NY, 10314, USA

    Sumit Mukherjee, Rahman Hussaini, Richard White, Doaa Atwi, Angela Fried & Probal Banerjee

  4. College of Arts and Science, New York University, New York, NY, 10003, USA

    Samay Sampat

  5. Department of Otolaryngology-Head and Neck Surgery, The Ohio State University Medical Center, Columbus, OH, 43210, USA

    Longzhu Piao & Quintin Pan

  6. Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, The Ohio State University Comprehensive Cancer Center, Columbus, OH, 43210, USA

    Longzhu Piao & Quintin Pan

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  1. Sumit Mukherjee
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Contributions

PB, SM, QP, and LP conceived and planned the experiments. SM, RH, RW, DA, AF, SS, and LP carried out the experiments. All the authors contributed to the interpretation of results. SM and PB took the lead in writing the manuscript. All authors provided critical feedback and helped in shaping the final manuscript.

Corresponding author

Correspondence to Probal Banerjee.

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Conflict of interest

Probal Banerjee has a pending patent application “Activity Enhancing Curcumin Compositions and Methods of Use”, PCT/US14/67819 pending. The remaining authors declare no conflict of interest.

Ethical approval and ethical standards

All procedures performed in studies involving animals were in accordance with the ethical standards of the Institutional Animal Care Committees (IACUC) at the College of Staten Island (approval # 11 − 008) and The Ohio State University Medical Center (approval # 2009A0172).

Animal source

Adult C57BL/6 female mice (2–6 months old) used for the TC-1 tumor experiments were bred and handled at the College of Staten Island (CUNY) [8]. Athymic nude (NCr) mice were purchased from Charles River Laboratories (Wilmington, MA) for the Head and Neck Squamous Cell Carcinoma (HNSCC) UMSCC47 tumor xenograft model and housed at The Ohio State University animal facility [7]. Both the strains mentioned above were bred and handled in accordance to the consent of respective Institutional Animal Care Committees (IACUC) at the College of Staten Island and The Ohio State University Medical Center. All animal protocols were approved by the respective Institutional Animal Care Committees (IACUC) at the College of Staten Island and The Ohio State University Medical Center.

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Mukherjee, S., Hussaini, R., White, R. et al. TriCurin, a synergistic formulation of curcumin, resveratrol, and epicatechin gallate, repolarizes tumor-associated macrophages and triggers an immune response to cause suppression of HPV+ tumors. Cancer Immunol Immunother 67, 761–774 (2018). https://doi.org/10.1007/s00262-018-2130-3

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