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Using curcumin to turn the innate immune system against cancer
Journal Pre-proofs Review Using Curcumin to Turn the Innate Immune System Against Cancer Sumit Mukherjee, Juliet N.E. Baidoo, Angela Fried, Probal Banerjee PII: DOI: Reference:
S0006-2952(20)30034-4 https://doi.org/10.1016/j.bcp.2020.113824 BCP 113824
To appear in:
Biochemical Pharmacology
Received Date: Accepted Date:
19 December 2019 22 January 2020
Please cite this article as: S. Mukherjee, J.N.E. Baidoo, A. Fried, P. Banerjee, Using Curcumin to Turn the Innate Immune System Against Cancer, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp. 2020.113824
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© 2020 Elsevier Inc. All rights reserved.
Using Curcumin to Turn the Innate Immune System Against Cancer
Sumit Mukherjee$*¶, Juliet N.E. Baidoo*, Angela Fried*, and Probal Banerjee$fl
Department of Chemistry$ and The Center for Developmental Neurosciencefl at The College of Staten Island, NY 10314. *Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York (CUNY), New York, NY 10016. ¶Current
address: Department of Cardiothoracic Surgery, Weill Cornell Medicine-New York
Presbyterian Hospital, NY 10065. Email address: [email protected]
Correspondences should be addressed to: Probal Banerjee, Ph.D., Professor, Department of Chemistry and the Center for Developmental Neuroscience, The City University of New York at The College of Staten Island, Staten Island, NY, 10314. Phone: (718) 982-3938. FAX (718) 9823944. Email addresses: [email protected]; [email protected] Running title: Using the innate immune cells in cancer therapy Number of figures: One
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Abstract: Curcumin has been at the center of vigorous research and major debate during the past decade. Inspired by its anti-inflammatory properties, many curcumin-based products are being sold now to manage various forms of arthritis. Parallel preclinical studies have established its role in dissolving beta-amyloid plaques, tau-based neurofibrillary tangles, and also alpha-synucleinlinked protein aggregates typically observed in Parkinson’s disease. In cancer research, most cancer cells in culture are eliminated by curcumin at an IC50 of 15-30 M, whereas the maximum in vivo curcumin concentration achieved in humans is only about 6 M. Additionally, a decade ago, no improvement over the placebo groups was observed in clinical studies using free curcumin as an anticancer agent. The lack of anticancer efficacy was attributed to its low bioavailability, which results from the low water-solubility and high metabolic rate in vivo. Newer lipid-complexed or antibody-targeted forms have been used and these studies have revealed an exciting property of curcumin, which involves repolarization of the tumor-promoting, tumorassociated microglia/macrophages (TAMs) into a tumoricidal form and recruitment of natural killer cells from the periphery. This review will cover some efforts to explore the effect of appropriatelydelivered curcumin to dramatically alter the tumor microenvironment, thereby launching an indirect attack on the tumor cells and the tumor stem cells.
Reviewing some aspects of
immunotherapy, this article will argue for the use of the innate immune cells in cancer therapy.
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1. Introduction: The spice component curcumin (CC) harbors selective cytotoxic activity against a wide range of cancer cells in culture. Since cancer research has been programmed for years to investigate the direct cytotoxic activity of agents against cancer cells, the initial surge of research on CC expected that, like most chemotherapeutic agents, it would eliminate cancer cells in vivo through direct killing. However, due to its poor solubility in water and rapid metabolism in vivo, CC per se displays very poor bioavailability in vivo. Consequently, a rude shock was encountered through inconclusive clinical trials, which seriously eroded interest in CC and, in fact, created a strong bias against it. In the midst of genome-wide profiling of mutations, promise of personalized cancer treatment, and the discovery of new synthetic drugs to combat the immunosuppressive effects of chemotherapy, CC was relegated to a “non-innovative” status by the major funding organizations to avoid “wasteful research” in the U.S. Thus, by expecting that CC would directly kill cancer cells just like the chemotherapeutic agents, a major property of CC has been overlooked: the ability of CC to stimulate the tumor-associated innate immune cells, which could be exploited to design safe immunotherapeutic agents.
2. The tumor microenvironment, a strength as well as the Achilles heel of the tumor: Could we use it to eliminate the tumor? After many years of effort to treat glioblastoma (GBM) brain tumor by surgical removal of the tumor followed by attempts to eliminate remaining cancer cells using radiation and chemotherapy, it has been realized that the quiescent GBM stem cells that grow in tumor niches are resistant to both chemo- and radiation therapies [1-4].
These cells express multi-drug-resistance and
antiapoptotic proteins and play a central role in the recurrence of GBM [1]. These cells are safely cradled in the tumor microenvironment, in which cancer cell-released factors such as colony stimulating factor-1 (CSF-1), CX3CL1, GDNF, GM-CSF cause recruitment and transformation of innate immune cells such as microglia and macrophages into an alternatively-activated pro-tumor
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state (named as M2-like) [5].
In this M2-like state, these tumor-associated microglia/
macrophages (TAMs) in turn secrete factors such as TGF-, IL-6, IL-10, and EGF, which promote cancer cell proliferation and metastasis [5]. For example, the TAM-released TGF- stimulate the GBM cells and GBM stem cells to secrete matrix metalloprotease-9 (MMP-9), which enables them to metastasize through tissue and blood vessels [5]. Furthermore, these M2-like TAMs harbor induced and activated signal transducer and activator of transcription 3 (STAT3), which triggers the synthesis of the enzyme arginase1 (ARG1). ARG1 cleaves the amino acid arginine, which is the precursor of the tumoricidal molecule nitric oxide (NO) [6]. Additionally, activated STAT3 also signals through IL10 to suppress the enzyme inducible nitric oxide synthase (iNOS) and the corresponding marker cytokine IL12 [7]. Radiation of glioma causes recruitment of the bone marrow-derived myeloid cells partly by triggering the binding of stromal cell-derived factor-1 (SDF1) to its receptor, CXCR4, thereby promoting vasculogenesis and tumor recurrence. In the prevailing approach of targeting single proteins with selective, synthetic inhibitors, it has been suggested that a clinically approved small molecule inhibitor of CXCR4 signaling, AMD3100, could improve the outcome of radiotherapy [8]. However, this approach was effective against vasculogenesis but not angiogenesis. Such effort to block downstream signaling molecules has been the major strategy in cancer therapy for a number of years. Many signaling molecules that are mutated in cancer cells have been targeted using synthetic drugs and a plethora of clinical trials have been conducted to test the life-prolonging effect of each drug in patients. However, we need to realize that no matter what drug is used to regulate a downstream effect, the TAMs, which are at the apex of most tumor-nurturing effects, will keep maintaining the tumor-promoting microenvironment.
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Findings discussed above establish that the tumor microenvironment acts as a nurturing armor for the cancer cells. So, we have asked “Could we design a strategy to turn the same tumor microenvironment, which functions as a “nurturing armor” for the cancer cells, into a state that is lethal to both cancer cells as well as cancer stem cells”? During the past five years, we have used a mouse model, created by orthotopic implantation of mouse GBM cells GL261 into syngeneic C57BL6 mice, to implement such a change in the GBM microenvironment [7, 9-11]. This change, which was brought about by appropriately-delivered CC, was associated with complete and long-term elimination of the GBM tumor and rescue of 50-60% of the GBM harboring mice. An important point needs to be considered here. Cell culture studies have revealed that CC per se eliminates most cancer cells in vitro with an IC50 of 15-30 M. However, in vivo CC concentrations by any delivery method never reach this level. Therefore, is it possible that CCmediated repolarization of the tumor microenvironment is achieved at a much lower concentration of CC? The possibility that low doses of CC may be effective in stimulating the immune system has been mentioned earlier [12]. The remaining sections of this review will elaborate on this important question.
3. Effort to increase bioavailability of curcumin: Despite the dampening of interest in CC as a chemotherapeutic agent, due to its efficacy as an anti-inflammatory agent against conditions such as rheumatoid arthritis [13], efforts to increase the bioavailability of CC have continued. Multiple strategies were soon used to boost CC’s plasma concentration and, more importantly, make it last for a longer time. One such strategy involved the preparation of phytosomal curcumin, which involved the formation of presumably a hydrogenbonded complex of curcumin with phosphatidylcholine. Using phytosomal curcumin, a curcuminglucuronate concentration of 2.76 M was achieved in mice and 0.38 M in human for a few hours
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[14, 15]. While these concentrations were far from sufficient to kill cancer cells directly, they could perhaps be adequate to dramatically alter the microenvironment within both peripheral as well as brain tumors. Among the many phytosomal versions of CC that are commercially available as supplements for joint health, we have tested one, Meriva [15], and standardized the dosage of this product for the rescue of GBM-harboring mice, and repolarization of TAMs from the tumorpromoting M2-like form to the tumoricidal M1-like phenotype [7, 9]. Besides this delivery form of CC, we have also tested a highly efficient, CD68 antibody (Ab)-linked curcumin prodrug that targets GBM cells, which we have demonstrated to express very high levels of CD68 [9, 16]. Once the Ab-CC adduct is endocytosed by the GBM cells, intracellular esterases release free CC, which then decimates the GBM cells at low nanomolar concentrations of antibody-linked CC [9]. We have successfully used this strategy with both melanoma- and GBM-targeted antibodies, but it can be used for any cancer cell that can be targeted with a selective antibody [9, 16, 17]. Since the microglia/macrophages (the TAMs) also express CD68 at a lower level, the adduct is also taken up by these cells, but instead of obliterating the TAMs, CC causes a dramatic repolarization of the TAMs from the M2-like to the M1-like state.
Our published mechanistic analysis is
discussed in the next section. Finally, we have also designed a liposomal form of CC and also a synergistic combination of CC with resveratrol and epicatechin gallate (TriCurin), which proved to be effective in causing repolarization of TAMs and elimination GBM tumors [10].
4. Attacking the tumor by manipulating the tumor microenvironment: As mentioned earlier, a CD68 Ab-CC adduct, phytosomal CC, and TriCurin, all afforded a dramatic switch in the TAMs (for GBM brain tumors) and macrophages (for peripheral tumors generated by subcutaneous implantation of TC-1 cervical cancer cells in C57BL8 mice) from the pro-tumor and immunosuppressive M2-like phenotype to the tumoricidal and pro-inflammatory M1 phenotype [9, 10, 18, 19]. Thus, the tumor-associated microglia/ macrophages could be manipulated to wield a Trojan Horse-like attack by the tumor-associated M1-like TAMs (Fig. 1).
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Additionally, the TAMs secrete the chemokine MCP-1 (a.k.a. CCL2), which in turn causes intratumor recruitment of activated natural killer (NK) cells and macrophages from the blood stream. This would cause further amplification of CC’s effect because the activated NK cells kill tumor cells and also secrete IFN, which binds to its receptor on microglia/macrophages to stabilize the M1 phenotype. Thus, while the M1-like microglia/macrophages secrete the cytotoxic molecule nitric oxide (NO) to kill tumor cells, the activated NK cells also eliminate cancer cells, cancer stem cells, and the resting macrophages (M0-type) by forming immune synapses with these cells [10, 19-22]. This is consistent with the report that the activated NK cells kill both M2 and M0-type macrophages, thus sparing the M1-like macrophages [23].
5. Repolarization of cytotoxic T cells and the microenvironment of the peripheral tumors: Adaptive immunity, which is defined by the presence of T or B lymphocytes, includes both CD8+ cytotoxic T (Tc) cells that directly kill the tumor cells, CD4+ helper T cells that regulate the functional activity of both T and B cells, and B cells that present antigen and produce antibodies [24]. Mice intraperitoneally injected with the murine mammary carcinoma cell line Ehrlich’s ascites carcinoma, revealed depletion of memory T cells, suppression of type 1 immune responses and augmentation of type 2 immune responses, and attenuated effector T cells (including the Tc cells) in the presence of tumors [25]. This was associated with increased Treg cells (which block Tc activation) and induced immunosuppressive cytokines, such as transforming growth factor- (TGF) and interleukin-10 (IL-10). Similar to our observation in the innate immune system, CC treatment brought about suppression of TGF- an IL-10, which in turn caused a downregulation of Treg cells. In effect, CC suppressed the type 2 immune responses and enhanced Tc cell population and type 1 immune responses [25].
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6. The adaptive immune system is used in immunotherapy: could the multi-targeted, naturally-occurring polyphenols function as broad-range-immune-boosting adjuvants? Immunotherapy broadly involves two approaches, both using the T-cells in the adaptive immune system [26]. The cancer cells express elevated levels of the programed cell death 1 pathway ligand (PD-L1), whereas the Tc cells express the receptor for this ligand PD-1. The binding of PD-L1 to PD-1 causes dampening of the cytotoxic activity of the Tc cells. The first approach (termed as immune check point inhibition) uses inhibitors of PD-L1 or PD-1 to suppress the activity of the pathway and, thereby, unmask the tumoricidal activity of the Tc cells.
The second
approach, named as chimeric antigen receptor T (CAR-T) cell therapy, includes genetically engineering a patient’s T cells to express surface receptor proteins that can recognize the antigens presented by the cancer cells in the patient. This triggers Tc cell mediated elimination of cancer cells. Both approaches are often combined with other strategies that further empower the Tc cells.
A few examples of such adjuvant strategies include cytotoxic T lymphocyte
associated protein (CTLA-4) blockade and COP9 signalosome 5 (CSN5) inhibition [26, 27]. Expressed constitutively by the Treg cells, CTLA-4 dampens the activity of the Tc cells [28]. Therefore, CTLA-4 inhibition yields an immune boosting effect in PD-L1-targeted therapy. CSN5, which is the fifth component of the COP9 signalosome complex, is responsible for deubiquitination and stabilization of PD-L1 and thereby, inhibition of T-cell mediated adaptive immune activity. Intriguingly, CC has been shown to inhibit CSN5, thus causing Tc-mediated elimination of cancer cells in a mouse model of breast cancer [27]. In addition to CSN5 inhibition, many other adjuvant therapies are currently in use along with PD-L1/PD-1 inhibition, such as radiation therapy, chemotherapy, and cytokine treatment [26].
7. Does immunotherapy have to be modified for the primary brain tumors such as GBM? The salient feature of current immunotherapy is that it uses the adaptive immune cells, which are supplied by the lymph nodes, spleen, and the bone marrow. In recent years, this adaptive immune
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system-based strategy of immunotherapy has brought immense hope and promise to cancer therapy. However, after a few years of application, it was revealed that immunotherapy often comes with serious side effects and is not effective in many cases [28]. For example, tumor cells from patients resistant to anti-PD-1 therapy appeared to have acquired mutations resulting in the loss of IFN response elements or MHC class I, which made them resistant to T cell-mediated elimination [29]. Such resistance could even involve the TAMs (macrophages). Thus, tumorassociated macrophages (the M2-like TAMs) removed the therapeutic antibody from the surface of the T cells in vivo, thereby making the anti-PD-1 therapy ineffective [30]. As for the application of immunotherapy to GBM, although a few studies have suggested that some of the immune check point inhibitors could cross the blood-brain-barrier [31], the first phase III study of PD pathway inhibition for GBM patients was unsuccessful to meet its primary goal [31-34]. Although adaptive immune cells enter the brain under specific conditions, generally, the sentries of the brain are not the adaptive immune cells but the innate immune cells. However, the strategy of employing the readily available innate immune system in cancer therapy has not been considered clinically. The immense advantage of using the innate immune cells like the microglia/macrophages is that they are already present in and around the tumors as tumorpromoting M2-like TAMs (Fig. 1). We have demonstrated this in both GBM brain tumors as well as TC-1 cervical cancer cell-evoked peripheral tumors in mice [7, 11]. Consequently, these TAMs can be repolarized into the M1 phenotype and NK cells recruited at any stage of tumor growth by some virtually non-toxic polyphenols such as CC, TriCurin, or Meriva. Using our orthotopic mouse model of GBM, we have shown this treatment to be effective for tumor elimination even at a stage when the tumor occupies about 8-10% (~50 l) of the brain volume (500-600 l) [9, 16, 17]. This is significantly larger than the volume of a human GBM brain tumor when it is surgically removed (the size of a golf ball with the average diameter of 2.15 cm and volume of 41.65 ml, which is about 3.5% of the human brain volume).
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8. Time to use the innate immune system in cancer therapy: CC in its native state, but not in appropriate delivery forms, had been used in some clinical trials for various cancers about a decade ago. When such clinical trials failed, all interest in the use of such polyphenols as anticancer agents or even as adjuvants was lost. We attribute this turn of events mainly to the fact that for a long time, the general strategy for cancer therapy has been to find agents that directly kill cancer cells. However, we have observed that such direct killers of cancer cells (chemotherapeutic agents) also kill the rapidly-dividing immune cells and are not effective against the relatively quiescent cancer stem cells [1-4]. During the last decade, better delivery forms of CC have been designed in order to stabilize this polyphenol in vivo. Although such delivery forms of CC were not able to achieve an in vivo concentration of CC that could directly kill the cancer cells, our research during this period has established that CC or its synergistic formulations like TriCurin can effectively eliminate cancer cells by repolarizing the TAMs and recruiting the activated NK cells into the tumor. Therefore, we emphasize here that the primary brain tumors are not expected to be bathed by lymph drainage of adaptive immune cells, but, as we have shown, the GBM tumor is filled with innate immune cells like microglia in the form of M2-like TAMs. Using the strategy of appropriately directing polyphenols like CC into the brain, we could turn the M2-like TAMs into M1-like and also have NK cell recruited to incite a cellular “mutiny” within the GBM tumor. Therefore, it is time to re-kindle our interest in the newer delivery forms of polyphenols, which can bolster cancer therapy by repolarizing the innate immune cells that populate most tumors.
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Figure legends: Fig. 1. Multi-target mechanism of activation and recruitment of innate immune cells by curcumin. CC in Curcumin Phytosome Meriva (CCP) [15] is known to suppress signal transducer and activator of transcription 3 (STAT3) in the tumor-associated microglia [35]. Since STAT3 is known to signal suppression of STAT1, the latter undergoes induction [36]. Induced (phosphorylated) P-STAT1 induces the synthesis of iNOS, IL12, thus increasing M1-like microglia [37, 38]. Released by M1-like TAM, the chemokine MCP-1 compromises the blood-brain barrier, gets released into blood, binds to its receptor (CCR2) on M1-like macrophages, and recruits them into the GBM microenvironment in the brain [39-44]. Concurrently, M1-like macrophages in blood cause STAT1-mediated IL12 synthesis and secretion [45]. These IL12 molecules bind to IL12 receptor (IL12R) expressed by the NK cells, which activates these cells to release interferongamma (IFN) release [46]. The secreted IFN molecules cause receptor-mediated inhibition of STAT3 in the macrophages [47-49], thus amplifying STAT1 induction and IL12 release [36]. Additionally, IFN also directly causes receptor-mediated stimulation of STAT1 [50, 51]. This stabilizes the M1-like phenotype and the activation of NK cells. Concomitantly, brain-released MCP-1 binds to CCR2 on the IL12-activated NK cells [52] and recruits them into the GBM microenvironment [40]. The activated NK cell recruits form immune-synapses with GBM cells and GBM stem cells, releasing perforin to lyse these targets [53, 54]. Additionally, the activated NK cells also kill resting (M0) microglia, thus enriching the M1-like microglia in the TAM [55]. Simultaneously, the M1-like macrophages and M1-like microglia within the GBM induce iNOSmediated nitric oxide (NO) release [42, 56], which eliminates GBM cells and GBM stem cells.
Author credit statement: Sumit Mukherjee: experimentation, writing, reviewing, and data curation. Juliet N.E. Baidoo: experimentation, data curation, and editing.
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Angela Fried: experimentation, reviewing and editing. Probal Banerjee: Supervision, conceptualization and writing.
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S0006-2952(20)30034-4 https://doi.org/10.1016/j.bcp.2020.113824 BCP 113824
To appear in:
Biochemical Pharmacology
Received Date: Accepted Date:
19 December 2019 22 January 2020
Please cite this article as: S. Mukherjee, J.N.E. Baidoo, A. Fried, P. Banerjee, Using Curcumin to Turn the Innate Immune System Against Cancer, Biochemical Pharmacology (2020), doi: https://doi.org/10.1016/j.bcp. 2020.113824
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© 2020 Elsevier Inc. All rights reserved.
Using Curcumin to Turn the Innate Immune System Against Cancer
Sumit Mukherjee$*¶, Juliet N.E. Baidoo*, Angela Fried*, and Probal Banerjee$fl
Department of Chemistry$ and The Center for Developmental Neurosciencefl at The College of Staten Island, NY 10314. *Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York (CUNY), New York, NY 10016. ¶Current
address: Department of Cardiothoracic Surgery, Weill Cornell Medicine-New York
Presbyterian Hospital, NY 10065. Email address: [email protected]
Correspondences should be addressed to: Probal Banerjee, Ph.D., Professor, Department of Chemistry and the Center for Developmental Neuroscience, The City University of New York at The College of Staten Island, Staten Island, NY, 10314. Phone: (718) 982-3938. FAX (718) 9823944. Email addresses: [email protected]; [email protected] Running title: Using the innate immune cells in cancer therapy Number of figures: One
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Abstract: Curcumin has been at the center of vigorous research and major debate during the past decade. Inspired by its anti-inflammatory properties, many curcumin-based products are being sold now to manage various forms of arthritis. Parallel preclinical studies have established its role in dissolving beta-amyloid plaques, tau-based neurofibrillary tangles, and also alpha-synucleinlinked protein aggregates typically observed in Parkinson’s disease. In cancer research, most cancer cells in culture are eliminated by curcumin at an IC50 of 15-30 M, whereas the maximum in vivo curcumin concentration achieved in humans is only about 6 M. Additionally, a decade ago, no improvement over the placebo groups was observed in clinical studies using free curcumin as an anticancer agent. The lack of anticancer efficacy was attributed to its low bioavailability, which results from the low water-solubility and high metabolic rate in vivo. Newer lipid-complexed or antibody-targeted forms have been used and these studies have revealed an exciting property of curcumin, which involves repolarization of the tumor-promoting, tumorassociated microglia/macrophages (TAMs) into a tumoricidal form and recruitment of natural killer cells from the periphery. This review will cover some efforts to explore the effect of appropriatelydelivered curcumin to dramatically alter the tumor microenvironment, thereby launching an indirect attack on the tumor cells and the tumor stem cells.
Reviewing some aspects of
immunotherapy, this article will argue for the use of the innate immune cells in cancer therapy.
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1. Introduction: The spice component curcumin (CC) harbors selective cytotoxic activity against a wide range of cancer cells in culture. Since cancer research has been programmed for years to investigate the direct cytotoxic activity of agents against cancer cells, the initial surge of research on CC expected that, like most chemotherapeutic agents, it would eliminate cancer cells in vivo through direct killing. However, due to its poor solubility in water and rapid metabolism in vivo, CC per se displays very poor bioavailability in vivo. Consequently, a rude shock was encountered through inconclusive clinical trials, which seriously eroded interest in CC and, in fact, created a strong bias against it. In the midst of genome-wide profiling of mutations, promise of personalized cancer treatment, and the discovery of new synthetic drugs to combat the immunosuppressive effects of chemotherapy, CC was relegated to a “non-innovative” status by the major funding organizations to avoid “wasteful research” in the U.S. Thus, by expecting that CC would directly kill cancer cells just like the chemotherapeutic agents, a major property of CC has been overlooked: the ability of CC to stimulate the tumor-associated innate immune cells, which could be exploited to design safe immunotherapeutic agents.
2. The tumor microenvironment, a strength as well as the Achilles heel of the tumor: Could we use it to eliminate the tumor? After many years of effort to treat glioblastoma (GBM) brain tumor by surgical removal of the tumor followed by attempts to eliminate remaining cancer cells using radiation and chemotherapy, it has been realized that the quiescent GBM stem cells that grow in tumor niches are resistant to both chemo- and radiation therapies [1-4].
These cells express multi-drug-resistance and
antiapoptotic proteins and play a central role in the recurrence of GBM [1]. These cells are safely cradled in the tumor microenvironment, in which cancer cell-released factors such as colony stimulating factor-1 (CSF-1), CX3CL1, GDNF, GM-CSF cause recruitment and transformation of innate immune cells such as microglia and macrophages into an alternatively-activated pro-tumor
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state (named as M2-like) [5].
In this M2-like state, these tumor-associated microglia/
macrophages (TAMs) in turn secrete factors such as TGF-, IL-6, IL-10, and EGF, which promote cancer cell proliferation and metastasis [5]. For example, the TAM-released TGF- stimulate the GBM cells and GBM stem cells to secrete matrix metalloprotease-9 (MMP-9), which enables them to metastasize through tissue and blood vessels [5]. Furthermore, these M2-like TAMs harbor induced and activated signal transducer and activator of transcription 3 (STAT3), which triggers the synthesis of the enzyme arginase1 (ARG1). ARG1 cleaves the amino acid arginine, which is the precursor of the tumoricidal molecule nitric oxide (NO) [6]. Additionally, activated STAT3 also signals through IL10 to suppress the enzyme inducible nitric oxide synthase (iNOS) and the corresponding marker cytokine IL12 [7]. Radiation of glioma causes recruitment of the bone marrow-derived myeloid cells partly by triggering the binding of stromal cell-derived factor-1 (SDF1) to its receptor, CXCR4, thereby promoting vasculogenesis and tumor recurrence. In the prevailing approach of targeting single proteins with selective, synthetic inhibitors, it has been suggested that a clinically approved small molecule inhibitor of CXCR4 signaling, AMD3100, could improve the outcome of radiotherapy [8]. However, this approach was effective against vasculogenesis but not angiogenesis. Such effort to block downstream signaling molecules has been the major strategy in cancer therapy for a number of years. Many signaling molecules that are mutated in cancer cells have been targeted using synthetic drugs and a plethora of clinical trials have been conducted to test the life-prolonging effect of each drug in patients. However, we need to realize that no matter what drug is used to regulate a downstream effect, the TAMs, which are at the apex of most tumor-nurturing effects, will keep maintaining the tumor-promoting microenvironment.
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Findings discussed above establish that the tumor microenvironment acts as a nurturing armor for the cancer cells. So, we have asked “Could we design a strategy to turn the same tumor microenvironment, which functions as a “nurturing armor” for the cancer cells, into a state that is lethal to both cancer cells as well as cancer stem cells”? During the past five years, we have used a mouse model, created by orthotopic implantation of mouse GBM cells GL261 into syngeneic C57BL6 mice, to implement such a change in the GBM microenvironment [7, 9-11]. This change, which was brought about by appropriately-delivered CC, was associated with complete and long-term elimination of the GBM tumor and rescue of 50-60% of the GBM harboring mice. An important point needs to be considered here. Cell culture studies have revealed that CC per se eliminates most cancer cells in vitro with an IC50 of 15-30 M. However, in vivo CC concentrations by any delivery method never reach this level. Therefore, is it possible that CCmediated repolarization of the tumor microenvironment is achieved at a much lower concentration of CC? The possibility that low doses of CC may be effective in stimulating the immune system has been mentioned earlier [12]. The remaining sections of this review will elaborate on this important question.
3. Effort to increase bioavailability of curcumin: Despite the dampening of interest in CC as a chemotherapeutic agent, due to its efficacy as an anti-inflammatory agent against conditions such as rheumatoid arthritis [13], efforts to increase the bioavailability of CC have continued. Multiple strategies were soon used to boost CC’s plasma concentration and, more importantly, make it last for a longer time. One such strategy involved the preparation of phytosomal curcumin, which involved the formation of presumably a hydrogenbonded complex of curcumin with phosphatidylcholine. Using phytosomal curcumin, a curcuminglucuronate concentration of 2.76 M was achieved in mice and 0.38 M in human for a few hours
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[14, 15]. While these concentrations were far from sufficient to kill cancer cells directly, they could perhaps be adequate to dramatically alter the microenvironment within both peripheral as well as brain tumors. Among the many phytosomal versions of CC that are commercially available as supplements for joint health, we have tested one, Meriva [15], and standardized the dosage of this product for the rescue of GBM-harboring mice, and repolarization of TAMs from the tumorpromoting M2-like form to the tumoricidal M1-like phenotype [7, 9]. Besides this delivery form of CC, we have also tested a highly efficient, CD68 antibody (Ab)-linked curcumin prodrug that targets GBM cells, which we have demonstrated to express very high levels of CD68 [9, 16]. Once the Ab-CC adduct is endocytosed by the GBM cells, intracellular esterases release free CC, which then decimates the GBM cells at low nanomolar concentrations of antibody-linked CC [9]. We have successfully used this strategy with both melanoma- and GBM-targeted antibodies, but it can be used for any cancer cell that can be targeted with a selective antibody [9, 16, 17]. Since the microglia/macrophages (the TAMs) also express CD68 at a lower level, the adduct is also taken up by these cells, but instead of obliterating the TAMs, CC causes a dramatic repolarization of the TAMs from the M2-like to the M1-like state.
Our published mechanistic analysis is
discussed in the next section. Finally, we have also designed a liposomal form of CC and also a synergistic combination of CC with resveratrol and epicatechin gallate (TriCurin), which proved to be effective in causing repolarization of TAMs and elimination GBM tumors [10].
4. Attacking the tumor by manipulating the tumor microenvironment: As mentioned earlier, a CD68 Ab-CC adduct, phytosomal CC, and TriCurin, all afforded a dramatic switch in the TAMs (for GBM brain tumors) and macrophages (for peripheral tumors generated by subcutaneous implantation of TC-1 cervical cancer cells in C57BL8 mice) from the pro-tumor and immunosuppressive M2-like phenotype to the tumoricidal and pro-inflammatory M1 phenotype [9, 10, 18, 19]. Thus, the tumor-associated microglia/ macrophages could be manipulated to wield a Trojan Horse-like attack by the tumor-associated M1-like TAMs (Fig. 1).
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Additionally, the TAMs secrete the chemokine MCP-1 (a.k.a. CCL2), which in turn causes intratumor recruitment of activated natural killer (NK) cells and macrophages from the blood stream. This would cause further amplification of CC’s effect because the activated NK cells kill tumor cells and also secrete IFN, which binds to its receptor on microglia/macrophages to stabilize the M1 phenotype. Thus, while the M1-like microglia/macrophages secrete the cytotoxic molecule nitric oxide (NO) to kill tumor cells, the activated NK cells also eliminate cancer cells, cancer stem cells, and the resting macrophages (M0-type) by forming immune synapses with these cells [10, 19-22]. This is consistent with the report that the activated NK cells kill both M2 and M0-type macrophages, thus sparing the M1-like macrophages [23].
5. Repolarization of cytotoxic T cells and the microenvironment of the peripheral tumors: Adaptive immunity, which is defined by the presence of T or B lymphocytes, includes both CD8+ cytotoxic T (Tc) cells that directly kill the tumor cells, CD4+ helper T cells that regulate the functional activity of both T and B cells, and B cells that present antigen and produce antibodies [24]. Mice intraperitoneally injected with the murine mammary carcinoma cell line Ehrlich’s ascites carcinoma, revealed depletion of memory T cells, suppression of type 1 immune responses and augmentation of type 2 immune responses, and attenuated effector T cells (including the Tc cells) in the presence of tumors [25]. This was associated with increased Treg cells (which block Tc activation) and induced immunosuppressive cytokines, such as transforming growth factor- (TGF) and interleukin-10 (IL-10). Similar to our observation in the innate immune system, CC treatment brought about suppression of TGF- an IL-10, which in turn caused a downregulation of Treg cells. In effect, CC suppressed the type 2 immune responses and enhanced Tc cell population and type 1 immune responses [25].
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6. The adaptive immune system is used in immunotherapy: could the multi-targeted, naturally-occurring polyphenols function as broad-range-immune-boosting adjuvants? Immunotherapy broadly involves two approaches, both using the T-cells in the adaptive immune system [26]. The cancer cells express elevated levels of the programed cell death 1 pathway ligand (PD-L1), whereas the Tc cells express the receptor for this ligand PD-1. The binding of PD-L1 to PD-1 causes dampening of the cytotoxic activity of the Tc cells. The first approach (termed as immune check point inhibition) uses inhibitors of PD-L1 or PD-1 to suppress the activity of the pathway and, thereby, unmask the tumoricidal activity of the Tc cells.
The second
approach, named as chimeric antigen receptor T (CAR-T) cell therapy, includes genetically engineering a patient’s T cells to express surface receptor proteins that can recognize the antigens presented by the cancer cells in the patient. This triggers Tc cell mediated elimination of cancer cells. Both approaches are often combined with other strategies that further empower the Tc cells.
A few examples of such adjuvant strategies include cytotoxic T lymphocyte
associated protein (CTLA-4) blockade and COP9 signalosome 5 (CSN5) inhibition [26, 27]. Expressed constitutively by the Treg cells, CTLA-4 dampens the activity of the Tc cells [28]. Therefore, CTLA-4 inhibition yields an immune boosting effect in PD-L1-targeted therapy. CSN5, which is the fifth component of the COP9 signalosome complex, is responsible for deubiquitination and stabilization of PD-L1 and thereby, inhibition of T-cell mediated adaptive immune activity. Intriguingly, CC has been shown to inhibit CSN5, thus causing Tc-mediated elimination of cancer cells in a mouse model of breast cancer [27]. In addition to CSN5 inhibition, many other adjuvant therapies are currently in use along with PD-L1/PD-1 inhibition, such as radiation therapy, chemotherapy, and cytokine treatment [26].
7. Does immunotherapy have to be modified for the primary brain tumors such as GBM? The salient feature of current immunotherapy is that it uses the adaptive immune cells, which are supplied by the lymph nodes, spleen, and the bone marrow. In recent years, this adaptive immune
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system-based strategy of immunotherapy has brought immense hope and promise to cancer therapy. However, after a few years of application, it was revealed that immunotherapy often comes with serious side effects and is not effective in many cases [28]. For example, tumor cells from patients resistant to anti-PD-1 therapy appeared to have acquired mutations resulting in the loss of IFN response elements or MHC class I, which made them resistant to T cell-mediated elimination [29]. Such resistance could even involve the TAMs (macrophages). Thus, tumorassociated macrophages (the M2-like TAMs) removed the therapeutic antibody from the surface of the T cells in vivo, thereby making the anti-PD-1 therapy ineffective [30]. As for the application of immunotherapy to GBM, although a few studies have suggested that some of the immune check point inhibitors could cross the blood-brain-barrier [31], the first phase III study of PD pathway inhibition for GBM patients was unsuccessful to meet its primary goal [31-34]. Although adaptive immune cells enter the brain under specific conditions, generally, the sentries of the brain are not the adaptive immune cells but the innate immune cells. However, the strategy of employing the readily available innate immune system in cancer therapy has not been considered clinically. The immense advantage of using the innate immune cells like the microglia/macrophages is that they are already present in and around the tumors as tumorpromoting M2-like TAMs (Fig. 1). We have demonstrated this in both GBM brain tumors as well as TC-1 cervical cancer cell-evoked peripheral tumors in mice [7, 11]. Consequently, these TAMs can be repolarized into the M1 phenotype and NK cells recruited at any stage of tumor growth by some virtually non-toxic polyphenols such as CC, TriCurin, or Meriva. Using our orthotopic mouse model of GBM, we have shown this treatment to be effective for tumor elimination even at a stage when the tumor occupies about 8-10% (~50 l) of the brain volume (500-600 l) [9, 16, 17]. This is significantly larger than the volume of a human GBM brain tumor when it is surgically removed (the size of a golf ball with the average diameter of 2.15 cm and volume of 41.65 ml, which is about 3.5% of the human brain volume).
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8. Time to use the innate immune system in cancer therapy: CC in its native state, but not in appropriate delivery forms, had been used in some clinical trials for various cancers about a decade ago. When such clinical trials failed, all interest in the use of such polyphenols as anticancer agents or even as adjuvants was lost. We attribute this turn of events mainly to the fact that for a long time, the general strategy for cancer therapy has been to find agents that directly kill cancer cells. However, we have observed that such direct killers of cancer cells (chemotherapeutic agents) also kill the rapidly-dividing immune cells and are not effective against the relatively quiescent cancer stem cells [1-4]. During the last decade, better delivery forms of CC have been designed in order to stabilize this polyphenol in vivo. Although such delivery forms of CC were not able to achieve an in vivo concentration of CC that could directly kill the cancer cells, our research during this period has established that CC or its synergistic formulations like TriCurin can effectively eliminate cancer cells by repolarizing the TAMs and recruiting the activated NK cells into the tumor. Therefore, we emphasize here that the primary brain tumors are not expected to be bathed by lymph drainage of adaptive immune cells, but, as we have shown, the GBM tumor is filled with innate immune cells like microglia in the form of M2-like TAMs. Using the strategy of appropriately directing polyphenols like CC into the brain, we could turn the M2-like TAMs into M1-like and also have NK cell recruited to incite a cellular “mutiny” within the GBM tumor. Therefore, it is time to re-kindle our interest in the newer delivery forms of polyphenols, which can bolster cancer therapy by repolarizing the innate immune cells that populate most tumors.
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9. References [1] W. Chen, Dong, J., Haiech, J., Kilhoffer, M.-C., and Zeniou, M., Cancer Stem Cell Quiescence and Plasticity as Major Challenges in Cancer Therapy, Stem Cells International 2016 (2016) Article ID 1740935. [2] S. Bao, Wu, Q,., McLendon, R.E., Hao, Y., Shi, Q., Hjelmeland, A.B., Dewhirst, M.W., Bigner, D.D., and Rich, J.N., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response, Nature 444 (2006) 756-760. [3] D. Hambardzumyan, Squatrito, M., and Holland, E.C., Radiation resistance and stem-like cells in brain tumors, Cancer Cell 10 (2006) 454-456. [4] A.M. Bleau, Hambardzumyan, D., Ozawa, T., Fomchenko, E., Huse, J.T., Brennan, C.W., and Holland, E.C., PTEN/PI3K/Akt Pathway Regulates the SidePopulation Phenotype and ABCG2 Activityin Glioma Tumor Stem-like Cells, Cell Stem Cell 4 (2009) 226-235. [5] D. Hambardzumyan, Gutmann, D.H., and Kettermann, J., The role of microglia and macrophages in glioma maintenance and progression, Nature Neuroscience 19 (2016) 20-27. [6] T. Hagemann, Biswas, S., Lawrence, T., Sica, A., and Lewis, C.E., Regulation of macrophage function in tumors: the multifaceted role of NF-kB, Blood 113 (2009) 3139-3146. [7] S. Mukherjee, Fried, A., Hussaini, R., White, R., Baidoo, J., Yalamanchi, S., and Banerjee, P. , Phytosomal curcumin causes natural killer cell-dependent repolarization of glioblastoma (GBM) tumor-associated microglia/macrophage and elimination of GBM and GBM stem cells, Journal of Experimental & Clinical Cancer Research 37 (2018) 168. [8] M. Kioi, Vogel, H., Schultz, G., Hoffman, R.M., Harsh, G.R., and Brown, J.M., Inhibition of vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation in mice, J. Clin. Invest. 120 (2010) 694-705. [9] S. Mukherjee, Baidoo, J., Fried, A., Atwi, D., Dolai, S., Boockvar, J., Symons, M., Ruggieri, R., Raja, K., and Banerjee, P., Curcumin changes the polarity of tumor-associated microglia and eliminates glioblastoma, International Journal of Cancer 139 (2016) 2838-2849. [10] S. Mukherjee, Baidoo, J.N.E., Sampat, S., Mancuso, A., David, L., Cohen, L.S., Zhou, S., and Banerjee, P., Liposomal TriCurin, A Synergistic Combination of Curcumin, Epicatechin Gallate and Resveratrol, Repolarizes Tumor-Associated Microglia/Macrophages, and Eliminates Glioblastoma (GBM) and GBM Stem Cells, Molecules 23(1) (2018) 201. [11] S. Mukherjee, Hussaini, R., White, R., Atwi, D., Fried, A., Sampat, S., Piao, L., Pan, Q., and Banerjee P., 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 (2018) 781-774. [12] L.I. Xinjian, and Xiacheng, L., Effect of curcumin immune function of mice, Journal of Huazhong University of Science and Technology [Med Sci] 25 (2005) 137-140. [13] F. Oliviero, Scanu, Z., Zamimudio-Cueva, Y., Puni, L., and Spinella, P., Anti-inflammatory effects of polyphenols in arthritis, J. Sci. Food Agric. 98 (2018) 1653-1659. [14] J. Cuomo, Appendino, G., Dem, A.S., Schneider, E., McKinnon, T.P., Brown, M.J., Togni, S., and Dixon, B.M., Comparative Absorption of a Standardized Curcuminoid Mixture and Its Lecithin Formulation, Journal of Natural Products 74 (2011) 664–669. [15] T.H. Marczylo, Verschoyle, R.D., Cooke, D.N., Morazzoni, P., Steward, W.P., and Gescher, A.J., Comparison of systemic availability of curcumin with that of curcumin formulated with phsphatidylcholine, Cancer Chemother Pharmacol 60 (2007) 171-177. [16] P. Langone, Debata, P.R., Inigo, J.D.R., Dolai, S., Mukherjee, S., Halat, P., Mastroianni, K., Curcio, G.M., Castellanos, M.R., Raja, K., and Banerjee, P., Coupling to a Glioblastoma-directed Antibody Potentiates Anti-tumor Activity of Curcumin, International Journal of Cancer 135 (2014) 710-719.
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Figure legends: Fig. 1. Multi-target mechanism of activation and recruitment of innate immune cells by curcumin. CC in Curcumin Phytosome Meriva (CCP) [15] is known to suppress signal transducer and activator of transcription 3 (STAT3) in the tumor-associated microglia [35]. Since STAT3 is known to signal suppression of STAT1, the latter undergoes induction [36]. Induced (phosphorylated) P-STAT1 induces the synthesis of iNOS, IL12, thus increasing M1-like microglia [37, 38]. Released by M1-like TAM, the chemokine MCP-1 compromises the blood-brain barrier, gets released into blood, binds to its receptor (CCR2) on M1-like macrophages, and recruits them into the GBM microenvironment in the brain [39-44]. Concurrently, M1-like macrophages in blood cause STAT1-mediated IL12 synthesis and secretion [45]. These IL12 molecules bind to IL12 receptor (IL12R) expressed by the NK cells, which activates these cells to release interferongamma (IFN) release [46]. The secreted IFN molecules cause receptor-mediated inhibition of STAT3 in the macrophages [47-49], thus amplifying STAT1 induction and IL12 release [36]. Additionally, IFN also directly causes receptor-mediated stimulation of STAT1 [50, 51]. This stabilizes the M1-like phenotype and the activation of NK cells. Concomitantly, brain-released MCP-1 binds to CCR2 on the IL12-activated NK cells [52] and recruits them into the GBM microenvironment [40]. The activated NK cell recruits form immune-synapses with GBM cells and GBM stem cells, releasing perforin to lyse these targets [53, 54]. Additionally, the activated NK cells also kill resting (M0) microglia, thus enriching the M1-like microglia in the TAM [55]. Simultaneously, the M1-like macrophages and M1-like microglia within the GBM induce iNOSmediated nitric oxide (NO) release [42, 56], which eliminates GBM cells and GBM stem cells.
Author credit statement: Sumit Mukherjee: experimentation, writing, reviewing, and data curation. Juliet N.E. Baidoo: experimentation, data curation, and editing.
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Angela Fried: experimentation, reviewing and editing. Probal Banerjee: Supervision, conceptualization and writing.
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