Copper-lowering agents as an adjuvant in chemotherapy.

Copper is an important element essential for metabolism and normal human body function. Although it is an essential element, related imbalance leads to toxic effects. Studies have proved that there is an increase in copper level in cancer cells. Evidences suggest the link between increase in copper levels and progression of various types of cancers. Different strategies have been utilized to decrease the level of copper in various types of cancer cells. However, it was observed that cell machinery involved in copper homeostasis plays critical factor in lowering copper levels in cancer cells. The outcomes of many monotherapies consisting copper-lowering agents for the treatment of different types of cancers showed that the inhibition of single factor is not sufficient to inhibit the growth of cancer cells. The combination of copper-lowering agent with chemotherapeutic agent showed synergistic effect. Interestingly, the presence of copper-lowering agent in such combinations significantly improved the efficacy of chemotherapeutic agent. The present work has focused on the discussion of outcomes of studies involving anti-copper agent and chemotherapeutic agent and related future strategies.

Introduction

Copper is one of the important trace elements. It is essential for lung elasticity, neuroendocrine function, neovascularization, adequate growth, metabolism, and cardiovascular integrity.[1] However, it has been observed that an imbalance in the copper levels is often related to several complications such as Wilson's disease.[2] The presence of higher copper levels in cancerous tissue assists in metastasis due to enhancement in angiogenesis and cell proliferation.[3] Copper is also responsible for causing cellular damage through alteration in the metabolic pathways, resulting in an increase of reactive oxygen species (ROS).[4] Hence, reducing the cellular copper levels for the treatment of different types of cancers is explored by many researchers. There are various methods available to measure the change in copper levels induced by copper-lowering drugs. One of the methods utilize serum ceruloplasmin (Cp) as an alternate measure is to determine the copper levels. The normal serum Cp levels in humans lie between 20 and 35 mg/dl, whereas the observed levels of Cp in cancer patients were 20–75 mg/dl. The reduction in copper levels (below normal) may cause clinical manifestations such as mild anemia, leukopenia, severe bone marrow depression, diarrhea, cardiac arrhythmias, peripheral neuropathy, and inhibition of epiphyseal bone growth in children. The major limitation in detecting copper levels is reduced levels of Cp below certain levels. It becomes an insensitive marker of copper levels below 5 mg/dl.[5] This clearly indicates that while developing strategies to reduce cellular copper levels to treat cancer the above factors should be taken into consideration [Figure 1].

Figure 1

Ceruplasmin levels in human (mg/dl)[5]

When anticopper drugs are used to treat cancer, the cell machinery which controls the copper homeostasis is a key factor responsible in the level of anticancer activity and related side effects. The outcomes of monotherapies involving copper-lowering agents for the treatment of various cancers are reviewed in the current article. Furthermore, cancer treatment strategies consisting of combination of anticancer drugs and copper-lowering agents are reviewed.

Monotherapy

Use of single anti-copper drug to treat cancer.

Tetrathiomolybdate

Brewer et al.[5] conducted first human trial of induction and maintenance of copper deficiency with tetrathiomolybdate (TM), as an antiangiogenic therapy for cancer. The rate of reduction of Cp levels was slow, but there were no significant side effects. The TM treatment was effective in early stage cancer but was ineffective in advanced cancer. TM treatment with meal resulted in nontoxic complex with copper, as it depletes from the body slowly. TM treatment before or after meal also resulted in lowering of copper levels. The study outcome indicated antiangiogenic effect with lowered copper levels which can be useful in the treatment of cancer.[6] TM showed antiangiogenic effect without producing toxicity in the patients having metastatic cancer.[5] In another clinical study involving patients with advanced kidney cancer, TM was given with meals. It was well tolerated and showed anticancer effects by lowering the copper levels.[7] Furthermore, the precise lowering of copper levels to the mid-range might has helped to meet the cellular requirements for the normal functioning and also to inhibit the angiogenic cytokine signalling. Antiangiogenic agents mostly target single angiogenic factor while there are dozens of proangiogenic factors. Hence, the inhibition of one angiogenic factor or its receptor is not sufficient because the tumors may utilize other factors.[8]

D-Penicillamine

The infiltrative growth of generally invasive 9 L gliosarcoma was inhibited by low copper diet and copper depletion by using D-Penicillamine (D-pen) in male Fischer rats.[9] The levels of serum copper elevated early and increased in proportion to tumor growth in wide range of tumors. There was normal level of serum copper in isolated tumor. It was also observed that the serum copper levels rise again after re-growth and extension of tumor.[10] The associated mechanism involves the generation of H2O2 and ROS when Cu (II) is reduced to Cu (I) by D-pen.[11] However, D-pen has limited intracellular delivery, high hydrophilicity, metal catalyzed oxidation, and rapid elimination.[12]

Trientine

Trientine was used as a substitute to pen in the case of patients who were intolerant to pen. Trientine promotes removal of copper from the body through urine. Mechanisms of action of trientine was as an antiangiogenic and apoptosis inducer when used as an anticancer agent.[131415] Limitations of trientine therapy include worsening of neurological symptoms, lupus like reactions, reversible sideroblastic anemia, etc.[16]

Zinc

The inhibition of human prostatic carcinoma cell growth was caused by zinc possibly due to apoptosis and induction of cell cycle arrest.[17] Zinc inhibits copper absorption by its inductive effect on zinc–thionein synthesis.[18] There was high concentration of intracellular zinc in the prostate gland which diminished during the development of prostate cancer (PCa). The objective of the study was to understand the effect of zinc in cancer. The results of the study indicated that Zncl2 inhibited the proliferation of androgen receptor (AR) - retaining PCa cells. While the same effect was not observed in AR-deficient PCa cells. The expression of AR in transgenic adenocarcinoma mouse prostate-C2 and LNCaP cells was down regulated by zinc. It was depended on dose as well as time.[19] Pyrithione zinc (PYZ), is a complex of zinc, and functions as a metal ionophore and enhances the concentration of the intracellular zinc ion. PYZ was the most efficacious cytotoxic agent observed through analyzing inhibition of cell proliferation and induction of apoptosis in oral squamous cell carcinoma cells in vitro. Retaining the optimal ZN2+ concentration was important for cell survival as both increased and decreased ZN2+ levels-induced apoptosis in a variety of cell types.[20]

Disulfiram

Disulfiram (DSF) belongs to the dithiocarbamate family; it is a copper-binding agent and an inhibitor of aldehyde dehydrogenase. It is a well-known agent used in the treatment alcoholism. Copper containing MDA-MB-231 cells were treated with DSF which resulted in proteasome inhibition and apoptosis. DSF formed complex with copper. The DSF-copper complex showed proteasome inhibitory effect and apoptotic cell death in human breast cancer cells. However, the DSF-copper complex did not show proteasome inhibitory effect and apoptotic cell death in normal/immortalized cells.[21]

Overall Limitations of Monotherapy

Cancer cell growth is supported by many cellular factors. Furthermore, the coordination between these factors does exist. Because of the involvement of many proangiogenic factors and angiogenic inhibitors potentially interacting, angiogenesis has turned out to be potentially complex area. Hence, monotherapies targeting angiogenesis, as a single factor has not yielded the desired results. The outcomes of many monotherapies targeting only single cellular factor have emphasized that there is a need of inhibition of cancer cell growth by targeting multiple cellular factors.[8]

Combination Anticancer Therapy

Many strategies have been used to inhibit multiple cellular factors to treat cancer. There are studies which showed promising outcomes involving anticopper drugs and chemotherapeutic agents [Table 1].[3]

Table 1

Summary of combinations

Combination	Reference
Cisplatin + TM	[34]
TM + doxorubicin	[22]
TM alone or in combination with doxorubicin, mitomycin C, fenretinide, or 5-fluorouracil	[24]
TM in combination with MAPK inhibitors (dabrafenib, encorafenib, vemurafenib, binimetinib, cobimetinib, and trametinib)	[23]
TM + irinotecan + 5-flurouracil + leucovorin	[25]
Trientine + carboplatin	[26]
Disulfiram + BTZ	[35]
Disulfiram + cisplatin + vinorelbine	[28]
Disulfiram + docetaxel + doxorubicin	[29]
Disulfiram + cisplatin	[30]
D-pen + PGA	[12]
D-pen + idarubicin	[31]
Zinc + doxorubicin	[32]

TM=Tetrathiomolybdate, MAPK=Mitogen-activated protein kinase, BTZ=Bortezomib, PGA=Poly-L-glutamic acid

Combination of Tetrathiomolybdate and Anticancer Drugs

Cisplatin is used for the treatment of various cancers.[33] However, it has limited therapeutic use because of side effects and increased resistance. It was observed that pretreatment of cells with elevated levels of copper reduced cisplatin uptake and enhanced resistance to cisplatin. The role of Ctrl and copper was examined using mouse model of human cervical cancer with the focus on cisplatin uptake and related response. TM with cisplatin showed synergistic antitumor effect. TM helped in increasing the concentration of cisplatin exclusively in tumor cells with simultaneous inhibition of tumor angiogenesis. Pretreatment of cultured human cervical and ovarian cancer cells with TM led to enhancement of cisplatin sensitivity and adducts in tumor cells. Further, TM was able to elevate cisplatin efficacy in highly metabolic tumors by reducing the bioavailable copper.[34] These promising evidences suggest the important role of TM co-administration with anticancer therapy to achieve synergism. TM also induced doxorubicin (dox) sensitivity in resistant tumor cell lines. The combination of TM and dox was found to be more effective at inducing apoptosis as compared to monotherapy.[22] TM increased sensitivity of ovarian cancer cells toward anticancer drugs dox, fenretinide, 5-fluorouracil, and mitomycin C. Furthermore, TM increased dox cytotoxicity along with modulation of key regulators of apoptosis. The well-known limitation for the use of dox is cardiac toxicity. It was reduced with the use of TM in preclinical assessments.[24] The hypothesis that TM inhibits the inflammatory process particularly at the activated-T-lymphocyte stage is well established.[22] Furthermore, targeting the mitogen-activated protein kinase (MAPK) pathway in melanoma by reducing Cu availability with the use of Cu chelators like TM and MAPK inhibitors (e.g., dabrafenib, encorafenib, vemurafenib, binimetinib, cobimetinib, and trametinib) might be a new strategy.[23] A pilot trial involving 24 patients with metastatic colorectal cancer (CRC) was conducted. Combination of TM with 5-flurouracil, leucovorin (IFL) and irinotecan was evaluated. TM inhibited angiogenesis. It was observed that the combination of IFL with TM was well tolerated along with maintenance of dose intensity of IFL. Overall, the use of TM can be considered safe with the combination chemotherapy used in the treatment of advanced CRC.[25]

Trientine in Combination with Anticancer Drugs

Trientine, a copper-lowering agent, was used in combination with carboplatin in the treatment of patients with advanced malignancies. Trientine helped in the enhancement of tolerance toward carboplatin treatment.[26] Trientine in combination with methotrexate inhibited angiogenesis with suppression of angiogenic factors in human colorectal carcinoma.[27]

Disulfiram in Combination with Anticancer Agents

There was reversal of bortezomib (BTZ) resistance and increase in related toxicity in MM cell line, when BTZ and DSF were used in combination. Inhibition of CuZnSOD activity by DSF was observed. It was observed that the upregulation of the enzymes CuZnSOD and H2O2 detoxifying enzyme glutathione peroxidase-1 was associated with BTZ resistance in MM cell lines.[35] DSF in combination with cisplatin and vinorelbine was assessed in a phase II b trial for the management of metastatic non-small cell lung cancer. It was well tolerated and prolonged the survival of the patients.[28] Human breast cancer cell lines MCF7/adr (multiply drug resistant), BT474 and MCF7/wt were used to study the effect of combinations of docetaxel and DSF. MTT assay was used for the measurement of Cytotoxic effect. Docetaxel, dox or epirubicin and DSF showed synergistic effect.[29] DSF enhances cytotoxic effects of cisplatin in five cell lines (e.g., A549, WI38, PC3, and MCF7) when given in combination. The enhanced cytotoxic action was found to be synergistic and might be due to their ability to induce an activating transcription factor 3 which is a regulator of cisplatin-induced cytotoxicity.[30]

D-penicillamine in Combination with other Anticancer Drugs

D-pen associated hydrophilicity, impermeability to the cell membrane and reactive thiol group are the challenges in terms of its delivery to cancer cells. It can be overcome by the formation of a conjugate of D-pen and poly-L-glutamic acid (PGA). Increase in cytotoxicity and intracellular ROS levels were observed in MDA-MB-468 (which is a human breast cancer cell line), HL-60, and murine leukemia cells (P388) treated with PGA-D-pen conjugate. The PGA-D-pen conjugation induces apoptosis. Furthermore, the conjugate effectively increased the survival of CD2F1 mice.[12] In another study, the combination of D-pen and idarubicin was found to enhance the therapeutic index by 2–3 fold higher than the monotherapy. The combination resulted in 89% tumor growth inhibition while it was only 60% with idarubicin. It was also observed that there was increase in the median survival of the mice model.[31]

Zinc in Combination with an Anticancer Drug Doxorubicin

Zinc oxide (ZnO) nanoparticles with dox penetrate more efficiently into drug sensitive as well as multidrug-resistant cancer cells in comparison to free dox. Furthermore, the combination ZnO/dox showed synergistic cytotoxic effects while monotherapy of Zno and dox failed show high cytotoxicity. ZnO prevented the formation of tumors by downregulation of CD 44.[32]

Conclusion

Anticancer therapies involving only copper-lowering agents or other anticancer monotherapies have limited therapeutic outcomes than their combinations. There are many reasons for this, but the major reason might be the inhibition of single factor. Hence, combination therapy involving copper-lowering agents with anticancer drugs could be an effective strategy as it targets multiple factors. Many copper-lowering agents and anticancer drugs showed synergistic effects. Copper-lowering agents have proved to enhance the properties of many anti-cancer drugs. However, copper homeostasis pathways play a very critical role which affects the efficacy of the anticancer drugs. Detection of copper levels is vital in patients undergoing anticancer therapy. This review will help in considering copper lowering agents as an adjuvant therapy with anticancer drugs. Hence, studies should be conducted to establish effective combination therapies involving copper-lowering agents and anticancer drugs with thorough consideration of copper homeostasis pathways.

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Conflicts of interest

There are no conflicts of interest.