Is Curcumin the Answer to Future Chemotherapy Cocktail?

The rise in cancer cases in recent years is an alarming situation worldwide. Despite the tremendous research and invention of new cancer therapies, the clinical outcomes are not always reassuring. Cancer cells could develop several evasive mechanisms for their survivability and render therapeutic failure. The continuous use of conventional cancer therapies leads to chemoresistance, and a higher dose of treatment results in even greater toxicities among cancer patients. Therefore, the search for an alternative treatment modality is crucial to break this viscous cycle. This paper explores the suitability of curcumin combination treatment with other cancer therapies to curb cancer growth. We provide a critical insight to the mechanisms of action of curcumin, its role in combination therapy in various cancers, along with the molecular targets involved. Curcumin combination treatments were found to enhance anticancer effects, mediated by the multitargeting of several signalling pathways by curcumin and the co-administered cancer therapies. The preclinical and clinical evidence in curcumin combination therapy is critically analysed, and the future research direction of curcumin combination therapy is discussed.


Introduction
Cancer has remained a serious threat to public health worldwide for centuries, and its prevalence is multiplying at a worrying rate. According to global cancer statistics (GLOBO-CAN) 2020, there will be about a 47% increase in cancer incidence in 2040 as compared to the estimated cases in 2020. The growing burden of cancer cases will likely correspond to the mortality rate among cancer patients, especially breast cancer and colorectal cancer [1]. With continuous collaborative research efforts between scientists and clinicians, multiple treatment modalities have been developed and improved throughout the years to control cancer aggravation, for instance, surgery, chemotherapy, radiotherapy and immunotherapy [2]. Fortunately, these treatment interventions have led to a decline in the mortality rate for cancer patients. However, not all cancer patients benefit completely from these treatments, owing to the difference in susceptibility to cancer treatment, heterogeneity of cancer, therapeutic resistance development and unprecedented side effects [3]. Hence, more initiatives are needed in the quest for more potent treatment modalities for a wide range of cancer patients to overcome the therapeutic obstacles.
Over the years, the concept of combination therapy, which relies on two or more therapeutic agents, has been introduced in the development of cancer treatment [4]. Compared to conventional single-agent therapies, combination therapy exhibits numerous benefits, such as targeting multiple oncogenic pathways, reducing high toxicity levels implicated by monotherapy, improving the magnitude of therapeutic responses and reducing the likelihood of therapeutic resistance [5]. These benefits allow for the greater suppression of cancer cells and reduce the risk of cancer recurrence, thus improving the patient's quality of life [4,6]. Lately, curcumin has gained a great deal of interest, attributed to its broad range of medicinal properties [7]. Intriguingly, curcumin exhibited countless anticancer properties, such as limiting cancer cell proliferation, promoting tumor cell death and preventing metastasis [7,8]. Besides, curcumin supplementation greatly relieves the patients from experiencing adverse effects caused by conventional therapies [6]. Hence, these properties pose great advantages to the development of curcumin combination therapy for cancer treatment.
This review focuses on the use of curcumin in combination therapy in various cancers. The evasive mechanisms developed by cancer cells in response to cancer therapy are discussed. Curcumin combination therapies used are reviewed in depth in each type of cancer in both preclinical and clinical studies. We also addressed how curcumin modulates a variety of molecular targets in cancer cells in the combination treatment, to provide an insight into the multitargeting effects of such treatment cocktails.

Evasive Mechanisms and Chemoresistance
Though the development of cancer therapy has achieved progressive milestones from conventional chemotherapeutic agents to the more advanced monoclonal antibodies and immunotherapy, inconsistencies and irresponsiveness towards the therapies were observed among the cancer patients receiving various cancer treatments, resulting in mixed outcomes among patients. Owing to the neoplastic characteristics, cancer cells are continuously evolving and adapting to the stress response induced by cancer therapies, developing numerous evasive mechanisms pertaining to the hallmark of cancer as their defensive strategies against chemotherapy [9]. Figure 1 summarizes the different evasive mechanisms by cancer cells.

Sustained Chronic Proliferation
Cancer is notorious for the sustained chronic proliferation behaviour in the body [9]. It is capable of neutralizing the cytotoxicity effects by profoundly accelerating cell proliferation to promote survival benefits. This uncontrolled cell growth is greatly manifested by the constitutive activation of several proliferation-related signalling pathways, notably transforming growth factor-β (TGF-β), phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mechanistic target of rapamycin (mTOR), epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathways in cancer cells [3,10]. Additionally, cancer cells induce aberrant cell growth through the dysregulation of the cell cycle and modulation of cell cycle-related proteins. Evading the cancer therapy, cancer cells usually instigate high activities of cyclin-dependent kinase (CDK), with the abnormal amplification of cyclin D, to accelerate the cancer cell proliferation [11,12]. Concurrently, the uncontrolled cell cycle progression is tightly associated with the decreased expression or loss of function of CDK inhibitors such as p21 and p27 [13]. Apart from its intrinsic mutation in cancer cells, tumor suppressor protein p53 is frequently downregulated in response to cancer therapy, thereby abolishing its anti-proliferative, anti-apoptosis and anti-metastatic activities [14].

Restricted Apoptotic Mechanism
Restricting the apoptotic mechanism is another strategy of cancer cells to override the killing effects induced by conventional therapies. As an orchestrated event in regulating physiological function, apoptosis mainly involves the extrinsic death receptor (DR) pathway and intrinsic mitochondrial pathway, which are tightly regulated by the balance of apoptotic proteins [15]. To maintain its immortality and metastatic behaviours, cancer cells have established several mechanisms to avoid apoptosis induction by cancer therapies [16]. As extensively reviewed in the literature, the fate of cell death is modulated by the balance of B-cell lymphoma 2 (Bcl-2) family proteins, which are composed of pro-apoptotic proteins, such as Bcl-2 homology 3 (BH3)-interacting domain death agonist (Bid), BH3-only Bcl-2interacting mediator of cell death (Bim), Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist/killer (Bak) and Bcl-2-interacting killer (Bik), and anti-apoptotic proteins, such as Bcl-2, B-cell lymphoma-extra-large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1), and Bcl-2-like protein 10 (Bcl2L10) [17]. The ratio of Bax/Bcl-2 is sometimes applied as the prognostic marker and susceptibility of a particular cancer treatment to cancer patients [18,19]. The overexpression of Bcl-2 is highly correlated with poor therapeutic efficacy among cancer patients [20]. Besides, the inhibitor of apoptosis (IAP) protein families, including cellular inhibitor of apoptosis protein 1 (cIAP1), cellular inhibitor of apoptosis protein 2 (cIAP2), X-linked inhibitor of apoptosis protein (XIAP) and survivin, are important determinants in the restriction of cancer cell death. These IAPs bind to the active site of caspases, thus limiting caspase activation and apoptosis induction [16]. Furthermore, the impairment of the extrinsic DR pathway was demonstrated by cancer cells to evade the apoptosis induced by chemotherapies. Apart from the downregulation of DRs, the reduced surface expression of DRs due to endocytosis was initiated by cancer cells, limiting the sensitivity of cancer cells towards drug treatments [21,22].

Increased Drug Efflux Function
The drug efflux mechanism is increased in cancer cells as a means to escape drug cytotoxicity effects [23]. The drug efflux function is governed by multidrug resistance protein (MDR), which belongs to the ATP binding cassette (ABC) transport families, notably P-glycoprotein (P-gp) (also known as multidrug resistance protein 1 (MDR1)), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) [24]. The overexpression of MDRs is found in a considerable number of cancers with both intrinsic and acquired resistance [25]. Furthermore, the upregulated expression of oncogenic kinases, such as MAPK and extracellular-signal-regulated kinase (ERK) signalling, caused the elevation of MDR expression in cancer cells, thus limiting the drug sensitivity. Apart

Curcumin and Its Medicinal Properties
Curcumin, also regarded as diferuloylmethane, is a yellow polyphenol extracted from the rhizome of the Curcuma longa (turmeric) plant, belonging to the Zingiberaceae family [40]. Indigenous in south-eastern and southern tropical Asia, curcumin is vastly utilized for food preservation, colouring and flavouring in daily activities [41]. Moreover, curcumin is traditionally applied for pain-relieving and wound healing effects. Commercial curcumin products contain approximately 77 % curcumin, 18 % demethoxycurcumin and 5 % bisdemethoxycurcumin [42]. Out of those curcuminoids, curcumin exhibits the most potent medicinal properties as compared to demethoxycurcumin and bisdemethoxycurcumin [41]. A growing body of evidence has demonstrated the benefits of curcumin in treating various diseases, including metabolic syndromes, hyperlipidaemia, inflammatory skin conditions, neurodegeneration and rheumatoid arthritis. These clinical benefits are attributed to the anti-inflammatory, anti-oxidant, and wound healing activities of curcumin [2,8,40]. Moreover, curcumin can impede pathogenic infections by exerting a broad spectrum of anti-bacterial, anti-fungal and anti-viral activities [43]. Alongside profound medicinal properties, curcumin is listed as a "generally recognized as safe (GRAS)" compound by the Food and Drug Administration (FDA), supporting its safety and tolerability when consumed by patients [7].
Enormous attention has given to the exploration of anticancer properties in curcumin. To date, curcumin has shown its anticancer benefits in numerous cancers such as breast cancer [44,45], colorectal cancer [46], lung cancer [47], pancreatic cancer [48] and prostate cancer [49]. In fact, these anticancer effects depicted by curcumin are highly associated with the modulation of several oncogenic signalling pathways, which are essential in cancer development. Curcumin constrains these oncogenic signalling pathways and further limits the downstream pro-tumorigenic activities. In vitro studies illustrated that curcumin treatment limited the proliferation and caused cell cycle arrest in HT-29 colon cancer cells and PLC/PRF/5 liver cancer cells via the inhibition of cyclin D1, with the downregulation of NF-κB and cyclooxygenase-2 (COX-2) signalling [50][51][52][53]. Concurrent with the upregulation of tumor suppressor gene p53, curcumin repressed the proliferative potential of cancer cells via the downregulation of PI3K/Akt/mTOR signalling [54][55][56]. Furthermore, it also impedes cancer cells' survival and suppresses their metastatic ability through the downregulation of EGFR pathways [57,58] and inhibition of MMP activities [59,60]. Apart from limiting the expression of IAP family proteins, curcumin promotes the apoptosis of cancer cells by increasing the expression of Bax while downregulating the expression of Bcl-2 in various cancer cells [61,62]. It has also been shown to be able to abrogate angiogenesis elicited by breast tumors via the suppression of VEGF [63]. The anticancer properties elucidated by curcumin are summarized in Figure 2.

Curcumin Combination Anticancer Therapy in Preclinical Studies
Conventional cancer therapies are challenged by the various defence mechanisms developed in cancer cells, hindering treatment success. Moreover, cancer cells often exhibit more than one mechanism of resistance, further complicating the treatment regimen. Hence, multiple targeting of these evasive mechanisms could potentially restore the sensitivity of cancer cells to cancer therapy, apart from eliminating the cancer cells completely. In this regard, curcumin holds a great promise in combination therapy to enhance the anticancer effects, while circumventing the problems encountered in conventional therapies. Since curcumin exhibits pleiotropic effects, the co-administration of curcumin in cancer therapy allows multiple targeting to the cancer-surviving and cancer-limiting mechanisms, while conventional monotherapy is restricted by single mechanism targeting only. Moreover, repetitive monotherapy caused the cancer cells to recruit other salvage pathways for survival benefits [4]. Hence, curcumin combination therapy could offset the evasion of cancer therapy and survivability of cancer cells, thereby overcoming the risk of cancer recurrence and treatment failure. To ensure the complete elimination of cancer cells, conventional monotherapy is often administered at a high dosage and causes a series of unwanted side effects [5]. Moreover, it was well known to be non-selective in killing proliferating cancer cells as well as healthy normal cells, upsetting the body immune system, and resulting in high toxicity [4]. These drawbacks could be alleviated by coadministering curcumin, such that a lower dosage of therapeutic agents is required. Thus, this could reduce the toxicity and adverse effects encountered by patients, besides yielding a significant therapeutic response [6,64]. To date, numerous preclinical investigations involving in vitro, in vivo and ex vivo studies have been conducted on the use of curcumin in combination therapy in various cancers (Table 1). Increased apoptotic activities via the activation of pro-apoptotic protein Bax; repression of anti-apoptotic protein Bcl-2; upregulation of caspase 3/8 activity.

3.
Reduction in cancer cell migration via the suppression of MMP-9 and ICAM-1.

4.
No toxicity to normal cell lines.

1.
Inhibition of tumor growth via the suppression of NF-κB.

2.
Prevention of breast cancer metastasis to lung tissues via the downregulation of MMP-9.

4.
Reduction in cancer cell migration and invasiveness via the suppression of MMP-9, CXCR4. 5.
Inhibition of autophagy via the downregulation of AMPK signalling.

2.
Increased apoptotic activities via the upregulation of caspase 3 activity; repression of anti-apoptotic protein Bcl-2.

3.
Inhibition of EMT via the downregulation of N-cadherin; upregulation of E-cadherin.

1.
Reduced tumor weight and volume via the suppression of Smad2 and Smad3 phosphorylation in tumor tissues.
Inhibition of cell proliferation.

2.
Increased endoplasmic reticulum stress-induced apoptotic activities via the activation of pro-apoptotic protein Bax; repression of anti-apoptotic protein Bcl-2; suppression of CHOP activity; induction of ROS production; upregulation of caspase 3/8 activity.

3.
Induction of S phase arrest.

4.
Reduced spheroid formation of cancer stem cells via the suppression of CD44, CD24 and EpCAM expression.

3.
Reduction in the angiogenesis effect by reduced expression of CD31 and Factor VIII in stained tissues.

3.
Induction of S phase arrest.

1.
Inhibition of tumor growth via the inhibition of mitotic index and increased apoptosis in tumor area.
Inhibition of cell proliferation via the suppression of Akt and ERK1/2 phosphorylation; induction of proteasomal-induced degradation of EGFR protein; inhibition of EGFR pathway.

2.
Increased apoptotic activities via the upregulation of caspase 3 activity and PARP cleavage; repression of anti-apoptotic protein survivin.

3.
Induction of autophagy-related cell death via the increase in LC3-II.
Inhibition of cancer cell migration.
Inhibition of cell proliferation via the suppression of EGFR expression.

2.
Inhibition of cancer cell migration and invasiveness.

3.
Sensitization to radiotherapy treatment via the suppression of EGFR expression.
Inhibition of cell proliferation via the upregulation of p21 and ERK1/2 phosphorylation; downregulation of NF-κB expression and Akt phosphorylation.

2.
Increased apoptotic activities via the activation of pro-apoptotic proteins Bax and p53; suppression of anti-apoptotic protein Bcl-2.

3.
Inhibition of cancer cell migration and invasiveness via the downregulation of MMP-2/9 activity.
Inhibition of cell proliferation.
Micelle system remained stable for 24 h.

3.
Harmless to normal cell line.

2.
Reduction in cardiotoxicity effect induced by doxorubicin via the downregulation of SOD and GSH-Px.

1.
Inhibition of cell proliferation via the suppression of Pgp function.

2.
Increased cellular uptake of doxorubicin and curcumin via endocytosis.

3.
Harmless to normal cell line.
Inhibition of cell proliferation.

2.
Increased cellular uptake of doxorubicin and curcumin.

2.
Increased apoptotic activities via the upregulation of caspase 3 and PARP cleavage.
Co-treatment was non-toxic to mice, especially to kidney, heart, liver and brain. [116,117] Tolfenamic acid 1.
Inhibition of cell proliferation.

2.
Increased apoptotic activities via the upregulation of caspase 3 activity, ROS production and PARP cleavage; repression of anti-apoptotic protein survivin.

3.
Induction of G0/G1 and G2/M arrest via the downregulation of NF-κB translocation to nucleus.
Inhibition of cell proliferation.

1.
Increased apoptotic activities via the upregulation of caspase 3 activity.

2.
Increased cytocidal effect of pancreatic cancer cells by NK cells via the production of IFN-γ.
[120] Increased apoptotic activities via the activation of pro-apoptotic protein Bak and Bid; repression of anti-apoptotic protein Bcl-2, Bcl-xL and Mcl-1. [121] Docetaxel (with curcumin encapsulated in EGFR peptide targeted, pH sensitive nanoparticle) 1. Nanoparticles remained stable and had better cumulative drug release in pH 5.0 as compared to pH 7.4.

2.
Increased cellular uptake of curcumin and docetaxel via EGFR-mediated endocytosis.
Inhibition of cell proliferation via the downregulation of mTOR and hTERT.

2.
Increased apoptotic activities via the activation of pro-apoptotic protein Bcl-xL; repression of anti-apoptotic protein Bcl-2.
Inhibition of cell proliferation via the downregulation of Akt, NF-κB and IκBα phosphorylation.

3.
Inhibition of angiogenesis via the downregulation of ERK signalling. Increased apoptotic activities via the activation of pro-apoptotic protein Bax; repression of anti-apoptotic protein Bcl-2.

4.
Inhibition of cancer cell migration.
Inhibition of tumor growth.
Inhibition of cell proliferation via the suppression of ATP activities; the release of ROS.

1.
Inhibition of tumor growth via the downregulation of Src, mTORC1 and STAT3 phosphorylation; suppression of AMPK activation.

2.
Decrease in tumor volume and weight of mice.
Inhibition of cell proliferation via the upregulation of p21 and p27.

2.
Increased apoptotic activities via the activation of pro-apoptotic protein Bax; upregulation of caspase 3 activity.

3.
Reversal of androgen deprivation therapy-induced chemoresistance via the upregulation of AR protein; downregulation of DNMT activity.

3.
Increased apoptotic activities via the repression of anti-apoptotic protein Bcl-2; activation of pro-apoptotic protein Bax; upregulation of PARP cleavage.

4.
Inhibition of cancer cell migration and invasiveness via the downregulation of VEGF and EGFR proteins.

5.
Inhibition of angiogenesis via the reduction in the number of HUVEC.

2.
Increased apoptotic activities via the elevation of Bax/Bcl-2 ratio.

3.
Inhibition of angiogenesis via the reduction in VEGF expression. [134] Gastric cancer Doxorubicin
Inhibition of cancer cell migration and invasiveness.
Inhibition of cell proliferation via the suppression of COX-2 and NF-κB.
Inhibition of cell proliferation.

2.
Increased apoptotic activities via the upregulation of DR5 and TRAIL/Apo2L.

1.
Inhibition of tumor growth via the suppression of NF-κB-related gene products such as cyclin D1 and c-Myc.

2.
Inhibition of tumor volume via the suppression of proliferation marker Ki67 and microvessel density marker CD31 and VEGF.

3.
Increased apoptotic activities via the suppression of anti-apoptotic protein Bcl-2 and survivin; upregulation of pro-apoptotic protein Bcl-xL.
Inhibition of cell proliferation via the upregulation of ERK and MEK phosphorylation; upregulation of p53 and p21; elevation of STAT3 phosphorylation.

2.
Increased apoptosis via the production of ROS; repression of anti-apoptotic protein Bcl-2 and XIAP.

3.
Inhibition of cancer cell migration.
Co-treatment was non-toxic to mice.
[139] Increased apoptosis via the repression of anti-apoptotic protein Bcl-2; activation of pro-apoptotic protein Bax.

3.
Harmless to normal cell lines.

1.
Inhibition of tumor growth via the suppression of BCR/ABL.

2.
Reduction in leukemia burden via the reduction in leukemic infiltration into the spleen. [140] Imatinib, vincristine 1.
Inhibition of cell proliferation via the suppression of VEGF and CCND1.

1.
Inhibition of cell proliferation via the downregulation of STAT3.

2.
Increased apoptosis via the repression of anti-apoptotic protein Bcl-xL.

Curcumin Combination Therapy in Breast Cancer
Compelling evidence has demonstrated the benefits of curcumin combination therapy as compared to monotherapy in breast cancer. As a selective estrogen receptor modulator, tamoxifen is renowned for the treatment of hormone-positive breast cancer [144]. Nonetheless, repeated treatments confer chemoresistance, attributed to the dysregulation of cell cycle and interruption on multiple signal transduction pathways [145]. An in vitro investigation reveals that the co-administration of curcumin and 4-hydroxytamoxifen (4-OHT), a metabolite of tamoxifen, could restore the sensitivity of 4-OHT of HR-positive MCF-7 cells through the downregulation of cyclin D1 and upregulation of p21. Concomitantly, the cell proliferative effect was reduced significantly via the repression of Akt/mTOR signalling pathways. Compared to either curcumin or 4-OHT alone, combined treatment also remarkably activated pro-apoptotic protein Bcl-xL and suppressed the Bcl-2 proteins, thereby further enhancing the apoptotic activities [65].
Apart from that, the Snail-related zinc-finger transcription factor (SLUG) overexpression, which is correlated to poor prognosis in various cancers [146,147], has been linked to tamoxifen resistance in breast cancer therapy [146]. The phenomenon was reversed with the combined treatment of curcumin and 4-OHT in MDA-MB-231 cells. Besides weakening mTOR activities, the reversal of chemoresistance was accompanied by enhanced mitochondrial-mediated apoptosis and the downregulation of hexokinase 2 (HK2) activities, therefore mediating cell death and preventing the metastatic behaviour of breast cancer cells, respectively [66].
Human epidermal growth factor receptor-2 (HER2) overexpression accounts for 15-30% of metastatic breast cancer, which exacerbates aberrant cell proliferation and cell survival in breast cancer patients [148]. To date, trastuzumab, an anti-HER2 monoclonal antibody, serves as the most efficacious targeted therapy in treating HER2-related breast cancer [149]. It was shown to be able to maximize its therapeutic potential when combined with other anticancer agents [150,151]. Through cell proliferation and cell cycle analysis, co-treatment of curcumin (10 µg/mL) and trastuzumab (10 µg/mL) significantly reduced cell proliferation and induced G2/M arrest in HER2-overexpressed BT-474 and SK-BR-3-hr (a herceptin resistant strain from SK-BR-3) breast cancer cells, compared to trastuzumab alone. This was accompanied by the suppression of HER2 expression with the inhibition of downstream targets such as NF-κB, Akt and MAPK signalling pathways. Further in vivo study revealed that BT-474 xenograft mice models had the smallest tumor volume after 4 weeks of curcumin (45 mg/kg) and trastuzumab (4 mg/kg) co-treatment, when compared to curcumin or trastuzumab alone [67].
Curcumin also serves as a potential adjuvant with other chemotherapeutic agents in augmenting anticancer effects. The combined treatment of curcumin and paclitaxel significantly suppressed the paclitaxel-mediated NF-κB expression and its regulatory genes COX-2, matrix metallopeptidase 9 (MMP-9), VEGF, and intercellular adhesion molecule 1 (ICAM-1), thus promoting the anti-proliferative and anti-metastatic behaviour in breast cancer cells [68,152]. Interestingly, further experiments proved that curcumin and paclitaxel curbed the metastasis of MDA-MB-435 breast cancer cells to lung tissues in xenograft mice models [69]. More importantly, this combination of curcumin (ranging from 25-225 mg/kg) and paclitaxel (5 mg/kg) was found to be safe and induced no toxicity effects in mice models [68].
Hindered by drug efflux and chemoresistance, doxorubicin was explored in combination with curcumin in breast cancer treatment [26]. A study reported that this co-treatment profoundly blocked the drug efflux function, as influenced by ATP binding cassette subfamily B member 4 (ABCB4) [72]. Additionally, the co-delivery of doxorubicin with curcumin loaded in solid lipid nanoparticles was shown to inhibit the tumor growth in mice by decreasing the P-gp surface expression besides increasing intracellular retention of doxorubicin. This has successfully augmented the cytotoxicity effect on breast cancer cells [73]. Besides, another study illustrated that curcumin inhibited the doxorubicin-induced EMT via the suppression of Akt, β-catenin and glycogen synthase kinase 3 β (GSK3β) pro-tein expression, emphasizing the importance of the combined treatment of curcumin and doxorubicin in inhibiting the metastasis of breast cancer cells [153].
Apart from the combination with chemotherapeutic agents, the combined treatment of curcumin with other natural compounds has also been investigated in breast cancer. Flow cytometry cell death analysis showed that the co-treatment of curcumin (5 µM) and berberine (25 µM) synergistically exerted apoptosis and autophagy cell death to MDA-MB-231 and MCF7 breast cancer cells [76]. Moreover, curcumin (1.5 µM) sensitized the AU565 breast cancer cells treated with quercetin (4 µM) and optiberry (2 µg/mL) to decrease lapatinib-mediated HER2 overexpression via the downregulation of HER2/Akt signalling pathways [74]. Another study reported the benefits of curcumin (200 mg/kg) and epigallocatechin gallate (EGCG) (25 mg/kg) in lowering the tumour burden of xenograft models via the reduction in phosphorylated Akt, EGFR and vascular endothelial growth factor receptor-1 (VEGFR-1) expression, highlighting the enhanced anticancer potential of this treatment regimen [75].

Curcumin Combination Therapy in Colorectal Cancer
To date, 5-fluorouracil (5-FU) remains one of the first-line treatments for colorectal cancer patients. Unfortunately, its clinical efficacy was limited by the low (about 10-15%) overall response in metastatic colorectal cancer patients [154]. Moreover, the toxicity experienced with increasing dosage of 5-FU has caused further constraints on the treatment [155]. Growing evidence has proven the role of curcumin in benefiting the chemotherapeutic efficacy of colorectal cancer preclinically. For instance, the toxicity profile was alleviated when the co-treatment of 5 µM curcumin lowered the concentration of 5-FU (0.01 nM; originally 10 nM) required in diminishing the cell proliferation of 3D cell culture models [77]. Concurrently, this was associated with G0/G1 cell cycle arrest via cyclin D1 inactivation [78,79]. Another cell cycle study showed that the co-treatment of curcumin and 5-FU caused S phase arrest in 5-FU-resistant HCT116 cell lines, implying that the regulation of cell cycle is cell-specific [80]. Furthermore, invasion assays revealed that this treatment combination reduced the migratory behaviour of colorectal cancer cells via the inhibition of MMP-9 and C-X-C chemokine receptor type 4 (CXCR4) expression, the downregulation of NF-κB expression, and disruption of ten-eleven translocation methylcytosine dioxygenase 1 (TET1)-naked cuticle homolog 2 (NKD2)-Wnt signalling pathways [77,79]. Apart from that, they also promoted the apoptotic activities in colon cancer cells via the upregulation of Bax and the cleavage of caspase 3, 8 and 9 [78,80]. Another study proved that 5-FU-induced autophagy was inhibited in HCT116 and HT29 colon cancer cells co-treated with curcumin and 5-FU, via the downregulation of Akt and mTOR activities [81]. The progression of 5-FU therapeutic resistance, either intrinsically or by repeated treatments, had exacerbated the uncontrolled cell division and survival of colorectal cancer cells via the elevation of prooncogenic NF-κB, Wnt and PI3K/proto-oncogene tyrosine-protein kinase (Src) signalling. However, this phenomenon was resolved when the colorectal cancer cells were co-treated with curcumin and 5-FU [77,79,80].
Other FDA-approved standard chemotherapeutic agents for colorectal cancer treatment include oxaliplatin, dasatinib and irinotecan [155]. Several preclinical studies demonstrated the improvement in therapeutic efficacy of colorectal cancer cells when these chemotherapeutic agents are co-administered with curcumin. In addition to increased growth inhibitory effects, the combined treatment of curcumin and other chemotherapeutic drugs, such as oxaliplatin or irinotecan, significantly promoted apoptotic activities in HT29 and HCT116 colon cancer cells via the increased reactive oxygen species (ROS) production and the upregulation of endoplasmic reticulum-associated protein C/EBP homologous protein (CHOP) [82,83]. Besides, the suppression of TGF-β, Smad-2 and N-cadherin indicated the EMT abolishment in colorectal cancer cells co-treated with curcumin and oxaliplatin [82,84,85]. The presence of CSCs in tumor microenvironment supports the notion of therapeutic failure and resistance. Therefore, targeting the CSCs can prevent cancer recurrence followed by tumor eradication [156]. The repeated administration of dasatinib and irinotecan has been shown to cause chemotherapeutic resistance in colorectal cancer cells. The co-administration of these drugs with curcumin had inhibited the sphereforming potential of colorectal CSC through the downregulation of CSC markers, such as CD44, CD133, epithelial cell adhesion molecule (EpCAM) and CD24, indicating that curcumin can alleviate the cancer progression by interrupting with CSCs activities when co-treated with other chemotherapy agents [84,85].
The combined treatment of multiple conventional chemotherapeutic agents has produced positive clinical outcomes in colorectal cancer patients to some extent. However, despite the improved overall survival and response rate initially, gradual irresponsiveness to repetitive treatment and modest toxicity were also experienced by the patients [155]. The combination of 5-FU and oxaliplatin were shown to elevate the expression of tumourpromoting proteins, such as EGFR, HER2, human epidermal growth factor receptor 3 (HER3), Akt and COX2, which rendered the cell survival and uncontrolled cell proliferation in chemo-surviving cells. Nevertheless, these tumor-promoting effects were curbed by the co-administration of curcumin [86,87]. Interestingly, the supplementation of curcumin increased the expression of insulin-like growth factor-binding protein 3 (IGFBP3) and promoted the binding between IGFBP3 and insulin-like growth factor 1 (IGF-1), sequestering the activation of IGF-1 and inhibiting the IGF-1/insulin-like growth factor type 1 receptor (IGF-1R) signalling, in 5-FU and oxaliplatin-treated colorectal cancer cells [86,87,94,95]. Furthermore, ex vivo spheroid cultures showed a great reduction in cell proliferation and the decreased expression of stem cell markers, such as Nanog, vascular endothelial growth factor receptor-2 (VEGFR-2) and octamer-binding transcription factor 4 (Oct4), with the combined treatment of 5-FU and oxaliplatin with curcumin. Further clinical studies have demonstrated that curcumin supplementation of 0.5-2 g/day to 5-FU-and oxaliplaitintreated patients were clinically safe [88]. Moreover, the co-administration of curcumin and 5-FU and oxaliplatin significantly suppressed EGFR signalling via the increased methylation status of EGFR, emphasizing the role of curcumin in the epigenetic modulation of colon cancer cells [89]. On the other hand, the combination of bevacizumab (0.4 mg/kg) and turmeric extract (with absorbable curcumin) (400 mg/kg) caused an inhibition of angiogenesis while promoting apoptosis in the tumor area in the xenograft model. Besides, the absence of systemic toxicity to the liver, kidney and heart in in vivo models signifies the safety and tolerability of this combined therapy [90]. Montgomery et al. (2016) reported that curcumin potentiated the anticancer effects of other natural compounds, where curcumin (12.5 µM) and silymarin (12.5 µM) synergistically augmented the apoptosis of DLD-1, HCT116 and LoVo colon cancer cells with increased caspase 3/7 activities, as compared to monotherapy [91]. Another study involving (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay and cell cycle analysis revealed that the co-treatment of curcumin (10 µM) and resveratrol (10 µM) showed synergistic anticancer effects in HCT116 colon cancer cells by inhibiting cell proliferation and inducing S phase arrest. This was associated with the reduction in oncogenic protein expression, notably EGFR, HER2, HER3 and IGF-1R levels. Further in vivo studies confirmed tumor growth reduction without any systemic toxicity, indicating the safety of curcumin and resveratrol [92].

Curcumin Combination Therapy in Lung Cancer
Current standard chemotherapeutic agents for non-small-cell lung carcinoma (NSCLC) patients are TKIs, such as gefitinib and erlotinib. Chemoresistance towards this treatment is becoming more prevalent, where the abnormal amplification of EGFR was massively identified in NSCLC patients [157]. Flow cytometric apoptotic analysis revealed that the co-treatment of curcumin (10 ng/mL) and gefitinib (0.1 mol/L) significantly augmented the apoptosis in NCI-H1975 lung cancer cells by blocking EGFR signalling pathways, notably Akt and ERK1/2 phosphorylation [93]. Apart from enhancing apoptosis, the co-treatment of curcumin and gefitinib induced autophagy-related cell death, which was associated with the suppression of histone deacetylase activities and the proteasomal degradation of EGFR proteins. Interestingly, in vivo studies confirmed a decrease in tumor weight of xenograft models as compared to control, through the reduced expression of oncogenic proteins EGFR, Akt and cyclin D1, and enhanced caspase 3/8 activities, while being harmless to other tissues in xenograft models [94,95]. Similar to gefitinib, erlotinib (1 µM) elevated its apoptotic effects to PC-9 lung cancer cells when co-administered with curcumin (25 µM) through the elevation of caspase 3 activities and the downregulation of EGFR proteins. In vivo studies further confirmed that the reduction of tumor growth was associated with the downregulation of NF-κB [96,97].
Platinum-based chemotherapy, such as cisplatin and carboplatin, forms the standard chemotherapy regimen in the advanced stage of lung cancer. Despite yielding better overall survival, these treatment regimens are often associated with undesirable toxicity and the relapse of lung cancer [158]. Studies on lung cancer cells showed that curcumin treatment could lower the concentration of cisplatin needed to achieve the same cytotoxicity effect when compared to cisplatin monotherapy [98,99]. This was supported by the reduction in cisplatin-induced thymidine phosphorylase (TP) and excision repair 1, endonuclease non-catalytic subunit (ERCC1)-related signalling such as PI3K/Akt/Snail signalling [100]. Additionally, the co-treatment of curcumin and cisplatin synergistically elevated apoptotic activities in A549 lung cancer cells, mainly via the upregulation of tumor suppressor proteins p53, p21 and downregulation of oncogenic proteins EGFR, HIF-1α, NF-κB, Akt, mTOR [99][100][101]. A transwell invasion study revealed that the co-treatment of curcumin and cisplatin diminished the invasiveness of lung CSCs that are the main drivers of tumor invasion and chemoresistance [102,103]. Likewise, the decrease in invasiveness was also observed when lung cancer cells were co-treated with curcumin (10 µM) and carboplatin (50 or 100 µM), through the repression of matrix metallopeptidase 2/9 (MMP-2/9) activities [104].
Other chemotherapeutic agents were also reported to have boosted anticancer effects when co-administered with curcumin. Cell survival assays demonstrated that curcumin (30 µM) enhanced the cytotoxicity of paclitaxel (30 µM) in paclitaxel-resistant A549 and H460 lung cancer cells through the suppression of microRNA-30c-5p-mediated metastasisassociated protein 1 (MTA1), which further limited the metastasis behaviour in lung cancer cells [105]. Besides, the co-delivery of curcumin and paclitaxel encapsulated in poly (B-cyclodextrintriazine) (PCDT) exerted the synergistic inhibition of clonogenic formation and increased apoptotic events in H1299 lung cancer cells, with better solubility, bioavailability and stability provided by the PCDT delivery system [106]. Doxorubicin, which is notorious for its cardiotoxicity, is also challenged by multidrug resistance development [107]. These problems were improved with a nanomicelle delivery system encapsulating both doxorubicin and curcumin, which was responsible for promoting endocytosis and increasing drug-release capacity, while being harmless to the normal cells in vitro and in vivo [107][108][109].

Curcumin Combination Therapy in Pancreatic Cancer
In pancreatic cancer, the co-treatment of curcumin and gemcitabine synergistically promoted apoptosis via the downregulation of NF-κB. Furthermore, the invasiveness of pancreatic cancer cells was diminished through the downregulation of N-cadherin, vimentin and the upregulation of E-cadherin [110]. Immunohistochemistry analysis indicated the decreased expression of EMT markers MMP-9, ICAM-1 and COX-2, suggesting a reduced metastasis behaviour in xenograft models co-treated with curcumin and gemcitabine. Furthermore, the inhibition of angiogenesis was observed in xenograft models via the suppressed expression of CD31 microvessel density marker [111]. Another study demonstrated that co-treatment of curcumin (20 µM) and gemcitabine (50 nM) remarkedly impeded the formation of spheroid-derived CSCs by inhibiting the polycomb repressive complex 2 (PRC2)/plasmacytoma variant translocation 1 (PVT1)/cellular-myelocytomatosis (c-Myc) axis [112]. The high level of multidrug resistance-associated protein 5 (MRP5) in pancreatic cancer cells was profoundly repressed by the co-treatment of curcumin and 5-FU, implying that curcumin potentiates the sensitivity of 5-FU to pancreatic cancer cells [114].
Lev-Ari et al. (2005) showed that the combined treatment of curcumin (15 µM) and celecoxib (25 µM) significantly reduced the cell proliferation and enhanced apoptotic effects in P-34 pancreatic cancer cells by suppressing COX-2 expression, as compared to celecoxib monotherapy [113]. The chemoprevention of curcumin (7.5 µM) and tolfenamic acid (50 µM) synergistically stimulated the apoptotic effects in pancreatic cancer cells via the downregulation of survivin and suppression of specificity protein 1 (Sp1). Additionally, this treatment regimen induced G1 and G2 cell cycle arrest with lesser translocation of NF-κB into the nucleus, implying a diminished cell proliferation [115].
In another study, cell death analysis illustrated that the combined treatment of curcumin (10 µM) and sulforaphane (5 µM) significantly enhanced the apoptotic effect of pancreatic cancer cells treated with aspirin (1 mM). With the decrease in cell survival, this chemoprevention regimen successfully impeded Akt phosphorylation and NF-κB activity [116]. Further maximizing the therapeutic efficacy, this co-treatment regimen was encapsulated in chitosan-coated solid lipid nanoparticles for better drug delivery in xenograft models. Apart from exhibiting no toxicity, this delivery system also demonstrated slow and sustained drug release profile, and thereby significantly lessened the tumor progression in in vivo studies [117,118]. On the other hand, apoptotic activity was synergistically augmented when BxPC-3 and Panc-1 pancreatic cancer cells co-treated with garcinol and curcumin showed elevated caspase 3/9 activities [119]. In a study investigating curcuminoids emulsified in omega-3 fatty acids combined with anti-oxidant Resolvin D1, it was revealed that the combination significantly induced apoptosis in pancreatic cancer cells via the activation of caspase 3 activity. Furthermore, this combined treatment potentiated the cytotoxicity effect and inhibited interferon γ (IFNγ) production in NK cells when co-cultured with pancreatic cancer cells [120].

Curcumin Combination Therapy in Prostate Cancer
Although the early stage of prostate cancer can be managed well with radiation and surgery, many patients eventually progress into metastatic prostate cancer due to irresponsiveness to androgen deprivation therapy (ADT) and chemotherapy resistance. This translates to the poor prognosis of prostate cancer patients [159,160]. Despite being approved by the FDA, the combination of docetaxel with prednisone or estramustine only demonstrated modest clinical benefits to prostate cancer patients [161]. The combined treatment of curcumin (20 µM) and docetaxel (10 nM) potentiated apoptotic effects in PC3 prostate cancer cells through the downregulation of Bcl-2, Bcl-xL, and Mcl-1 and upregulation of Bak and Bid. Furthermore, the decrease in cell proliferation was correlated with the reduced CDK1, Akt, EGFR, and HER2 expression [121]. An EGFR-targeted nanoparticle delivery system containing curcumin (0.58 µM) and docetaxel (0.058 µM) was developed to induce EGFR-mediated endocytosis in prostate cancer cells [162]. Besides being stable, this delivery system also successfully reduced the tumor burden of xenograft models without causing any systemic toxicity [122].
In recent years, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has become an attractive therapeutic agent in combating cancer through apoptosis. Current evidence showed limited positive outcomes in several clinical studies, mainly due to its poor agonist activity [163]. Despite being proven to be safe and cause no toxicity to normal tissues, TRAIL was profoundly correlated with its therapeutic resistance in cancer cells [22,164]. Therefore, a sensitizer plays an important role in overcoming TRAIL resistance. Curcumin has been shown to be able to sensitize TRAIL-resistant prostate cancer cells to TRAIL through the upregulation of death receptors death receptor 4 (DR4), death receptor 5 (DR5) and inhibited angiogenesis [164]. Similarly, another flow cytometry cell death analysis demonstrated that the combination of curcumin (10 or 25 µmol/L) and TRAIL (20 ng/mL) remarkedly induced apoptotic activities in LNCaP prostate cancer cells by downregulating NF-κB and suppressing nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (IκBα) phosphorylation [125]. Another in vivo experiment exhibited that the co-treatment of curcumin (50 mg/kg) and TRAIL (3 mg/kg) caused a significant reduction in tumor burden through downregulating Akt and NF-κB expressions [126]. Interestingly, these reports highlighted no toxicity effect in preclinical models, further proving the potential of curcumin and TRAIL combination as a safe and tolerable alternative treatment for prostate cancer in future [125,126].
The work by Eslami et al. (2020) showed that the combination treatment of curcumin (25 µM) and metformin (4 mM) synergistically elevated apoptotic activities with the downregulation of mTOR activities in LNCaP prostate cancer cells [123]. In terms of combination with other natural compounds, curcumin (50 µM) and epigallocatechin gallate (EGCG) (100 µM) collectively inhibited the proliferation of PC3 prostate cancer cells by causing G2/M and S phase arrest, which was linked to an elevation of p21 protein and reduced phosphorylation of retinoblastoma (Rb) [127]. Another study involving cell death analysis demonstrated that the supplementation of curcumin (5 to 10 µM) with arctigenin (1 µM) and EGCG (40 µM) synergistically induced apoptosis in LNCaP prostate cancer cells without affecting normal epithelial cells. The apoptotic effect was enhanced by an elevation of Bax/Bcl-2 ratio and a reduction in Akt and signal transducer and activator of transcription 3 (STAT3) phosphorylation [128]. Additionally, the co-administration of curcumin (60 mg/kg) and resveratrol (5.7 mg/mL) ameliorated the tumor burden of prostate cancer xenograft models via an elevated expression of tumour suppressor proteins and anti-oxidant activities [129]. Similarly, the co-treatment of curcumin with resveratrol and ursolic acid, respectively, worked in synergy to reduce the tumour volume of xenograft models, along with the decrease in mammalian target of rapamycin complex 1 (mTORC1) and signal transducer and activator of transcription (STAT) activities [130]. The combined treatment of curcumin (8.9 µM) and quercetin (8.9 µM) reversed the hypermethylation status of androgen receptor (AR) proteins that conferred ADT resistance, by restraining the activities of DNA methyltransferase. Hence, this led to enhanced AR-mediated apoptosis in prostate cancer cells [131].

Curcumin Combination Therapy in Other Cancers
Curcumin synergism for cancer treatment has also been studied in various other cancers. For instance, the co-treatment of curcumin (4.32 µmol/L) and 5-FU (2.16 µmol/L) exhibited a synergistic effect on the anti-proliferation of HepG2 hepatocellular carcinoma cells via the inhibition of NF-κB translocation from cytoplasm to nucleus. Concurrently, this co-treatment also suppressed the expression of COX-2 protein, thereby disrupting the uncontrolled cell survival [132]. In another study, the combined administration of curcumin (13 µM) and celecoxib (42.8 µM) synergistically induced apoptosis in liver cancer cells by elevating caspase 3 activity. Cell proliferation assay revealed that HepG2 hepatocellular carcinoma cells also exhibited a great reduction in the expression of cell survival proteins, such as Akt, NF-κB p65 and malondialdehyde (MDA), and the inhibition of VEGF expression, implying the potentiation of anti-proliferative and anti-angiogenesis effects by curcumin and celecoxib co-treatment [133].  tested the combination of curcumin (10 µM) and metformin (10 mM), and found that it induced a synergistic anti-proliferative effect on HepG2 hepatocellular carcinoma cells, without harming any normal cell lines. This anti-proliferative effect was attributed to the reexpression of tumor suppressor protein phosphatase and tensin homolog (PTEN) and p53. In vivo studies further confirmed that the co-treatment enhanced apoptosis activities via the upregulation of Bax/Bcl-2 ratio and elicited anti-angiogenesis effect via the downregulation of VEGF expression [134].
Although the incidence has declined steadily in the last few decades, gastric cancer still constitutes a global health issue as concerned by a low median survival rate of less than 12 months in gastric cancer patients [165]. Numerous studies suggested the potential of combination therapy to improve the clinical outcome of gastric cancer. Doxorubicin or etoposide repeated treatments led to the aberrant amplification of NF-κB, resulting in therapeutic resistance in gastric cancer cells. This problem was resolved by the pretreatment of curcumin (40 µmol/L) followed by doxorubicin (0.3 µmol/L) or etoposide (20 µmol/L) administration [136]. Similarly, curcumin and doxorubicin co-treatment showed significantly more induction of apoptosis and anti-mobility behaviour of AGS gastric cancer cells as compared to monotherapy and the untreated control [135]. In vivo studies also highlighted the benefits of curcumin (74 mg/kg) and 5-FU (52 mg/kg) coadministration in slowing down the tumor growth via the reduced expression of NF-κB and COX-2, without causing any toxicity effects in other body parts of gastric cancer xenograft models [137].
Being the gold standard intravesical immunotherapy for bladder cancer, Bacillus Calmette-Guerin (BCG) treatment is shown to drive therapeutic resistance in bladder cancer cells with continuous use [166]. Curcumin (10 µmol/L) has been shown to surmount this obstacle by elevating TRAIL and DR5 expressions, and downregulating NF-κB expression when being co-treated with BCG (106 colony-forming unit (CFU)), thus enhancing extrinsic apoptotic pathways and reversing BCG therapeutic resistance in bladder cancer cells. In vivo studies further elucidated the enhancement of anticancer effects by curcumin and BCG, by inhibiting cell proliferation via the downregulation of cyclin D1 and c-Myc, suppressing angiogenesis via the suppression of VEGF, and boosting apoptosis via the downregulation of Bcl-2 and survivin in xenograft models [138]. Furthermore, the co-treatment of curcumin (10 µM) and cisplatin (10 µM) induced ROS-mediated apoptosis, which was linked to the overactivation of MAPK/ERK kinase (MEK) and ERK phosphorylation. Moreover, curcumin and cisplatin co-treatment collectively elevated tumor suppressor protein PTEN as well as p53, and downregulated the phosphorylation of STAT3 in 253J-Bv and T24 bladder cancer cells [139].
In acute lymphoblastic leukemia, curcumin (15 µM) enhanced the apoptotic effects induced by imatinib (1 µM) on SUP-B15 cells through the downregulation of the Akt/mTOR pathway and the upregulation of the Bax/Bcl-2 ratio. Furthermore, the combination treatment also inhibited the expression of breakpoint cluster region protein-acute promyelocytic leukemia (BCR/ABL), in which imatinib monotherapy was unable to do so. In vivo studies verified that the combination of curcumin (25 mg/kg) and imatinib (5 mg/kg) reduced the leukemia burden in mice with a decreased expression of BCR/ABL [140]. Further investigations illustrated that curcumin (10 µM) with other chemotherapeutic agents, notably imatinib and vincristine, synergistically induced apoptosis via the downregulation of Bcl-2 and anti-angiogenesis effect via the downregulation of VEGF in ALL cells. Besides, combination treatment reversed the NF-κB activity induced by imatinib and vincristine [141]. Similarly, curcumin (40 µM) potentiated the inhibitory effect of thalidomide (80 µM) in acute myeloid leukemia cells KG-1 and U937 by downregulating the Bcl-xL expression in apoptosis and repressing STAT3 expression [142]. Apart from that, the combination of natural products, curcumin (13.47 µM) and quercetin (53.89 µM) synergistically induced apoptosis in chronic myeloid leukemia cells K562 via the downregulation of Bcl-2 and elevated cytochrome c release to the cytosol. Furthermore, the enhancement of apoptotic effects was evident through the elevation of ROS production and loss of mitochondrial membrane potential [143].

Curcumin Combination Therapy from Bench to Bedside
Successful preclinical results may not always translate to positive clinical outcomes; hence, clinical investigations in humans are crucial. To date, numerous clinical trials on curcumin combination therapy have been carried out (Table 2), evaluating the safety and tolerability of combined treatments, toxicity profiles, and therapeutic response of patients. These studies provide imperative information for clinicians in designing newly improved robust therapeutic interventions.

2.
The efficacy was maintained for more than 3 months in curcumin group, with higher RECIST score than baseline.

4.
TEAEs were lessreported in curcumin group as compared to placebo group. [167] NA Phase I dose escalation trial of docetaxel plus curcumin in patients with advanced and metastatic breast cancer 1 Breast cancer Docetaxel (100 mg/m 2 ) as intravenous infusion on day 1 of each 3 week cycle for 6 cycles. Premedicated with 50 mg BID of oral methylprednisolone given two days before and after chemotherapy. Six dose levels of curcumin (500 mg/day) for consecutive 7 days at each cycle.

1.
Maximal tolerated dose of curcumin was at 8000 mg/day.

2.
Out of 8 patients, 5 patients had PR and 3 patients had SD.

3.
Tumor marker CEA decreased significantly in patients with PR and SD from the 3rd cycle of treatment.

4.
VEGF significantly decreased by 30% between baseline and cycle no. 3, and by 21% between baseline and cycle no. 6. [168] NA Effect of imatinib therapy with and without turmeric powder on nitric oxide levels in chronic myeloid leukemia NA Chronic myeloid leukemia Curcumin group: imatinib (400 mg twice a day) along with turmeric powder (5 g three times/day dissolved in 150 mL of milk to improve its platability and absorption) for 6 weeks. Imatinib group: imatinib (400 mg twice a day for 6 weeks).

1.
Curcumin group achieved larger percentage of complete remission, with no significant difference with imatinib group.

3.
Limited common side effects were observed.
[169] To determine the safety, pharmacokinetics and effectiveness of irinotecan when given in combination with curcumin in patients with metastatic colorectal cancer.

2.
To better understand the interaction between curcumin and irinotecan by measuring levels of irinotecan in blood when a patient also takes curcumin.

3.
Information will result in improved dosing guidelines and lead to more effective treatment with lesser toxicity. Oral curcumin with 8 g was safe and feasible in patients with advanced pancreatic cancer.

NA
[173] The proportion of patients with PSA progression during the active curcumin treatment period (6 months) was significantly lower in the curcumin group than the placebo group.

3.
Curcumin was well tolerated and safe.
[176] Patients received curcumin (3 g) or placebo since 1 week before onset of radiotherapy until completion of their radiotherapy. External beam radiotherapy was given as daily fraction of 2 Gy to achieve a total dose of 74 Gy (5 times a week for about 8 weeks).

1.
Curcumin supplementation did not cause any side effects.

2.
In curcumin group, plasma total antioxidant capacity was significantly increased, and the activity of superoxide dismutase decreased after radiotherapy as compared to baseline level and placebo group.
[177] Curcumin combination therapy was proven to be safe and tolerable in the clinical trials of breast cancers [167], chronic myeloid leukemia [169], colorectal cancer [88,170], pancreatic cancer [172,174] and prostate cancers [176,177]. Furthermore, patients experienced lesser toxicity effects under the curcumin combination therapy with an improved quality of life. In a phase II placebo-controlled clinical trial, metastatic breast cancer patients encountered lesser treatment-emergent adverse events (TEAEs) under the treatment of paclitaxel (80 mg/m 2 ) and curcumin (300 mg solution) as compared to the patients receiving the placebo [167]. Moreover, the safety and tolerability of curcumin (up to 2 g) was well documented when co-administered with folinic acid, 5-FU and oxaliplatin (FOLFOX) chemotherapy to metastatic colorectal cancer patients [88,170]. In another randomized, double-blinded and placebo-controlled study, prostate cancer patients who received curcumin (1440 mg/day) until the completion of ADT did not experience any serious adverse effects [176]. Similarly, the supplementation of curcumin (3 g) did not result in any adverse effects to the prostate cancer patients throughout the radiotherapy [177]. These studies present the evidence of the safety and tolerability of curcumin when co-administered with conventional therapy.
In assessing the treatment response of curcumin combination therapy, a phase I doseescalation clinical trial showed that the maximal tolerated dose of curcumin (8000 mg/day) co-treated with docetaxel (100 mg/m 2 ) recorded 5/8 patients had partial response (PR) and 3/8 patients had stable disease (SD), with a significant decrease in the tumor marker carcinoembryonic antigen (CEA) and VEGF biomarkers of metastatic breast cancer [168]. Imatinib (400 mg twice per day) supplemented with turmeric powder (5 g three times/day dissolved in 150 mL of milk) achieved a higher complete remission in chronic myeloid leukemia patients. Additionally, curcumin combination treatment caused a better reduction in the nitric oxide level in patients, when compared with imatinib monotherapy [169].
In terms of the overall response rate (ORR), advanced breast cancer patients receiving curcumin (300 mg solution) and paclitaxel (80 mg/m 2 ) experienced significantly higher ORR than patients receiving the placebo [167]. The co-administration of curcumin (2 g) into FOLFOX-based chemotherapy showed a higher ORR (53.3%), with a longer median progression-free survival (PFS) and overall survival (OS), as compared to the ORR of FOLFOX-based monotherapy (11.1%) in metastatic colorectal cancer patients [170]. Another clinical trial of colorectal cancer patients receiving a daily dose of curcumin (2 g) with FOLFOX chemotherapy showed 91.7% ORR, with a median PFS of 34 weeks [88]. In a phase II study on the castration-resistant prostate cancer patients treated with docetaxel (75 mg/m 2 ), prednisone (5 mg) and curcumin (6000 mg/day), the ORR was 100%, with 40% having PR and 60% having SD, with a median time to progression of prostate-specific antigen (PSA) of 5.8 months [175]. Similarly, prostate cancer patients who received curcumin (1440 mg/day) in ADT had significantly lower PSA progression than patients who received ADT alone only [176]. There was an increase in plasma antioxidant capacity in prostate cancer patients who received curcumin (3 g) throughout the radiotherapy [177]. These studies demonstrated that curcumin showed treatment response when co-administrated with conventional cancer therapies.
Despite the remarkable outcomes discussed above, reports on curcumin supplementation exhibiting modest treatment response in pancreatic cancer patients have been found. In a phase II study with advanced pancreatic cancer patients treated with curcumin (8 g) and gemcitabine (1000 mg/m 2 ), the ORR was reported to be 45.5%, while 54.5% of the patients had tumor progression. Besides, some patients experienced gastrointestinal toxicity, such as abdominal fullness and pain [171]. In another phase II trial, advanced pancreatic cancer patients receiving curcumin (2000 mg/die) co-treated with gemcitabine (10 mg/m 2 or 1000 mg/mq) had about 61% of ORR; albeit, curcumin was safe and tolerable [172,174]. In another phase I/II study, gemcitabine-resistant pancreatic cancer patients who received curcumin (8 g) and gemcitabine (1000 mg/m 2 ) experienced a poor 1-year survival rate [173].
Current findings confirm the effectiveness of curcumin combination treatment in breast cancer, colorectal cancer, and prostate cancer, and less significant effectiveness in pancreatic cancer. Nevertheless, a few trials on pancreatic cancers are still ongoing, with no results available to date. Hence, the conclusion can only be drawn when more data are available. At this point of time, the evidence suggests that certain cancers and/or certain cell lines are more responsive to curcumin combination anticancer treatment. This is not surprising given the different characteristics of the cancer cells, the complexities of molecular pathways involved and the variety of treatment cocktails used. Numerous clinical studies on curcumin co-administration with various conventional cancer therapy are still in progress at the time of writing (Table 3).

1.
To determine the safety and feasibility of curcumin in chemotherapy.

2.
To assess changes in health-related QoL.

3.
To evaluate anti-inflammatory properties via CRP measure.

Research Gap and Future Directions
A large body of research demonstrated the role of curcumin in augmenting anticancer effects via combination therapy. Although the majority of studies reported positive therapeutic outcomes, some opposing results were observed in which the curcumin did not elicit a synergistic effect with combination therapy in various dosages [67,112,133]. The inconsistencies among the results warrant further investigations. To obtain robust methodology and ensure accurate results, careful considerations in the study planning is vital in both preclinical and clinical investigations.
In some studies, in vitro results were used to draw the conclusion without validation from in vivo findings. Care should be taken in interpreting these results, as in vitro findings can sometimes be a poor predictor of in vivo outcomes. Moreover, various factors, including cell-immune regulations in microenvironment and toxicity profiling, remain elusive without the integration of in vivo studies [178]. Therefore, in vivo investigations are necessary to validate the in vitro results before proceeding to human studies [41]. This means more laborious works are needed to be put in place to conduct the studies. However, it does more good than harm in the long run, with more solid preclinical evidence out there for progression into clinical trials.
Besides inducing apoptosis, curcumin is a potent regulator of autophagy in cancers. As a stress-coping mechanism, autophagy eliminates the cancer cells in the early stage of cancers. However, autophagy has also been shown to promote the survival of cancer cells by mitigating nutrient deprivation and hypoxia stress in the advanced stage of cancer [179,180]. There have been controversial opinions on whether curcuminmediated autophagy modulates pro-survival or pro-death mechanisms in cancer cells [181]. The combination treatment of curcumin (5 µM) and berberine (25 µM) synergistically induce autophagy-related cell death in MDA-MB-231 and MCF7 breast cancer cells via the increased activation of c-Jun N-terminal kinase (JNK) signalling [76]. Similarly, the enhancement of H157 and H1299 lung cancer cell death by the combination of curcumin and gefitinib was autophagy-dependent [95]. However, Kantara et al. (2014) demonstrated that curcumin promoted autophagic cell survival of doublecortin-like kinase 1 (DCLK1) positive-CSCs in colon cancer. The ablation of DCLK1 in colon cancer CSC only restored the cell death-promoting effects mediated by curcumin, highlighting that curcumin-mediated autophagy is dependent on the expression of DCLK1 [182]. Hence, curcumin-mediated autophagy can be a double-edged sword, and this depends on cancer types and targeted signalling pathways [183,184]. This presents room for investigation to further explore the crosstalk between apoptosis and autophagy mediated by curcumin combination treatment.
Albeit curcumin is safe and tolerable [7], and the toxicity issue, especially in persistent consumption, is often overlooked due to the lack of long-term clinical studies [185]. Moreover, increasing the dosage of curcumin has been shown to negatively regulate the anti-oxidant effects by inducing DNA damage and degrading p53 proteins, leading to potential carcinogenic effects [186,187]. In vivo studies demonstrated that curcumin is an active iron chelator. This implicates that the constitutive consumption of iron will impair the iron homeostasis in patients who have a suboptimal level of iron [188]. Several in vitro studies also illustrated that curcumin and other chemotherapeutic agents exhibited antagonistic effects, reversing the anticancer effects by restricting apoptosis and cytotoxicity potential in cancer cells [189]. For instance, the curcumin dietary supplementation antagonized the cyclophosphamide-induced tumor suppression in BT474 xenograft mouse models via the downregulation of JNK activities [190]. Besides, the co-treatment of curcumin and etoposide had an antagonistic effect in MC7 breast cancer cells, HepG2 liver cancer cells, HCT116 colon cancer cells and HeLa cervical cancer cells. Cell cycle analysis revealed that this co-treatment limited the cell death of cancer cells by restricting the cells entering M phase [191]. These issues warrant the need for stringent dose-response evaluation, attentive choices of drug combination, and long-term clinical follow up to determine the optimum dose of curcumin in maximizing therapeutic response without instigating any toxicity events.
Due to the heterogeneity of cancer and disparity in the genetic makeup of cancer patients, there could be discrepancies in the therapeutic efficacy of curcumin combination therapy among cancer patients. To address this issue, precision medicine can be applied to understand the underlying cause of cancer in a patient. This can be achieved through a multitude of drug screenings using curcumin as a main therapeutic agent combined with an array of conventional therapeutic agents. In addition to that, gene sequencing technology can be a useful tool in identifying tumor-associated vulnerabilities as therapeutic targets in cancer patients. Once a specific gene mutation is confirmed, the anticancer treatment can then be designed and tailored for the patient. Undoubtly, these interventions will foster the developments of highly effective curcumin combination therapy regimens that will target the precise cause of disease in an individual patient [192,193].
It is noteworthy that in most of the clinical trials, a remarkably high dose of curcumin was used. Curcumin is notorious for its hydrophobic nature, thereby restricting its pharmacokinetic potential by having poor aqueous solubility, poor bioavailability, and rapid systemic elimination from the body [194]. These characteristics have impeded its ability to reach the target area. As a result, a high dose of curcumin has to be administered to account for these shortcomings, in order to maintain the effective plasma concentration in the blood. In reality, a high dose of curcumin might be impractical in a clinical setting, as it necessitates the administration of several large tablets/capsules for a single dose. Moreover, the gastrointestinal side effect observed in one of the clinical trials signifies the potential irritant effect of large dose of curcumin. Hence, room for improvement exists in delivering curcumin as combination therapy for cancer patients. Specifically, this issue should be addressed before this treatment option is made available to patients. In this regard, refining the formulation of curcumin appears to be an important step. The improved formulation of curcumin should address the poor bioavailability and rapid degradation issues. The nanoformulation of curcumin is one of the options to be used, as these have been shown to be able to increase drug payload in a single dose, at the same time allowing the flexibility of modifying the nanoformulation according to needs. With the proper choice of excipients, the stability of curcumin could also be improved, where the nanomaterials could protect the compound from degradation. In fact, several reports illustrated that the application of nanoformulation in curcumin combination therapy has successfully demonstrated the enhanced aqueous solubility for better delivery [195], reversing multidrug resistance [108] and ensuring the distinctive biodistribution of therapeutic agents [107,196]. On the other hand, the nanoformulation of curcumin could also be designed to target a specific site of the body, to deliver an even higher dose of the compound to the site of action, instead of distributing to other organs. One of the examples is curcumin nanoparticles with a mucoadhesive effect for colorectal cancer [196][197][198]. It is expected that the nanoparticles would have prolonged contact time with the colon due to mucoadhesion between the nanoparticles and the colonic mucin. This will allow a greater amount of curcumin to be released on-site and act on the cancer cells. Utilizing the same concept, a nanoformulation containing both curcumin and the chemotherapeutic agent can then be engineered for such delivery for better treatment outcomes. It would be interesting to observe the outcome of the ongoing clinical trials on the combination chemotherapy with curcumin nanoformulations.

Conclusions
The search for an effective cancer therapeutic strategy remains a great challenge for the scientific community due to various side effects, general cytotoxicity to cancer and normal cells, and the development of therapeutic resistance, which often lead to therapeutic failure. Combination therapy presents a good option to alleviate the tumor burdens by lowering toxicity and simultaneously targeting multiple mechanisms that modulate tumor development, without harming the healthy cells.
Curcumin holds a great promise in the development of combination therapy, which is frequently paired with conventional chemotherapeutic drugs as well as other natural compounds. An enormous body of preclinical and clinical evidence had entailed the potential of curcumin in preventing the exacerbation of cancer development by modulating multiple signalling pathways when combined with other therapeutic agents. Apart from diminishing cell survival and elevating cancer cell death, curcumin combination therapy has a robust effect in alleviating the hallmarks of cancer, such as metastasis and angiogenesis progression. The combined treatment of curcumin with other conventional therapy could overcome the pitfalls contributed by persistent conventional treatments and the genetic constitution of cancer cells, resulting in the improvement of therapeutic outcome. However, the poor pharmacokinetic issues of curcumin need to be addressed first. This could be resolved through the application of appropriate drug delivery systems, such as nanoformulation, that could effectively improve the delivery of curcumin to the target sites. The controversy surrounding the use of curcumin, such as its antagonistic effect, autophagy modulation, and potential toxicity associated with its long term use, warrants more studies and long-term clinical monitoring. With the continuous exploration of curcumin combination therapy and a deep understanding of its modulation in anticancer mechanisms, curcumin combination therapy holds great promise as a new therapeutic approach in combating cancer, especially in cancer patients irresponsive to single conventional chemotherapy. It is noteworthy that deep molecular profiling is essential for understanding the tumor-associated biomarkers that are crucially influenced by the action of curcumin, thereby shaping the future combined therapeutic strategies that eventually will translate into better oncologic outcome.