Multitargeting Strategy Using Tetrathiomolybdate and Lenvatinib: Maximizing Anti-Angiogenesis Activity in a Preclinical Liver Cancer Model

Purpose To investigate the suppressing tumor-promoting effects via multi-anti-angiogenesis activity of the copper chelator (Ammonium Tetrathiomolybdate, TM) combined with lenvatinib for hepatocellular carcinoma. Material and methods Fifty-five C57 mice were injected subcutaneously with Hepa1-6 hepatoma cell suspensions into the right posterior thigh. Seven days later, all subcutaneous tumors were formed and the mice were randomly distributed into 5 groups: TM Group (G1), Lenvatinib Group (G2), TM+Lenvatinib Group (G3), Control Group (G4), and Copper (II) Gluconate Group (G5). And copper concentrations in serum and tumors were measured at the predetermined times. After fourteen days of treatments, tumor weight and volumes were analyzed, histology was observed, and the expressions of VEGF and microvessel density (MVD) in tumor tissues were measured by immunohistochemistry (IHC). Results Average concentration of copper in serum was 405.14 ug/L, 480.44 ug/L, and 679.80 ug/L in normal mice, in mice on 7 days after implantation, and in the control group, respectively. Similarly, intratumoral copper concentrations were greater in G4 mice (1511.90 ug/L) than mice on 7 days after implantation (852.80 ug/L) (p < 0.05). And the serum concentration of copper was 363.65 ug/L, 508.83 ug/L, 370.52 ug/L, 822.12 ug/L in G1, G2, G3, and G5 [G5 vs other Groups, all p < 0.05; (G1, G2, and G3) vs G4, p < 0.05; G1 vs G2, p = 0.013; G2 vs G3, p = 0.018; G1 vs G3, p = 0.903] while intratumoral copper concentrations was 674.31 ug/L, 988.91 ug/L, 550.52 ug/L, and 3004.95 ug/L in G1, G2, G3, and G5 And the average tumor weight was 0.55 g, 0.44 g, 0.08 g, 1.37 g, 3.11 g in the mice of G1, G2, G3, G4, and G5, respectively. [G5 vs other Groups, all p < 0.05; (G1, G2, and G3) vs G4, p < 0.05; G1 vs G3, p < 0.05; G2 vs G3, p < 0.05; G1 vs G2, p > 0.05]. Furthermore, tumors were collected for HE staining and IHC examination. The expression levels of VEGF in G1, G2, and G3 were 43.75, 32.48, and 15, and all of them were significantly lower than those in G4 (64.28) and G5 (89.03) (G4 vs G5, p < 0.05). Similarly, the trend of MVD was just like that of VEGF in the five groups whereas no significant difference occurred in G1 and G2. Conclusion The study shows that there is a significant positive correlation between tumor load and copper. Administration of copper can promote tumor progression, and copper chelating could suppress tumor growth. The combination of TM with lenvatinib can reduce tumor angiogenesis and improved the effect of antitumor treatment. These findings offer basic data support and theoretical foundation for the clinical application of the combination therapy.

Targeting the Copper Transport System to Improve Treatment Efficacies of Platinum-Containing Drugs in Cancer Chemotherapy

The platinum (Pt)-containing antitumor drugs including cisplatin (cis-diamminedichloroplatinum II, cDDP), carboplatin, and oxaliplatin, have been the mainstay of cancer chemotherapy. These drugs are effective in treating many human malignancies. The major cell-killing target of Pt drugs is DNA. Recent findings underscored the important roles of Pt drug transport system in cancer therapy. While many mechanisms have been proposed for Pt-drug transport, the high-affinity copper transporter (hCtr1), Cu chaperone (Atox1), and Cu exporters (ATP7A and ATP7B) are also involved in cDDP transport, highlighting Cu homeostasis regulation in Pt-based cancer therapy. It was demonstrated that by reducing cellular Cu bioavailable levels by Cu chelators, hCtr1 is transcriptionally upregulated by transcription factor Sp1, which binds the promoters of Sp1 and hCtr1. In contrast, elevated Cu poisons Sp1, resulting in suppression of hCtr1 and Sp1, constituting the Cu-Sp1-hCtr1 mutually regulatory loop. Clinical investigations using copper chelator (trientine) in carboplatin treatment have been conducted for overcoming Pt drug resistance due in part to defective transport. While results are encouraging, future development may include targeting multiple steps in Cu transport system for improving the efficacies of Pt-based cancer chemotherapy. The focus of this review is to delineate the mechanistic interrelationships between Cu homeostasis regulation and antitumor efficacy of Pt drugs.

Dehydroflavonolignans from Silymarin Potentiate Transition Metal Toxicity In Vitro but Are Protective for Isolated Erythrocytes Ex Vivo

2,3-Dehydrosilybin (DHS) was previously shown to chelate and reduce both copper and iron ions. In this study, similar experiments with 2,3-dehydrosilychristin (DHSCH) showed that this congener of DHS also chelates and reduces both metals. Statistical analysis pointed to some differences between both compounds: in general, DHS appeared to be a more potent iron and copper chelator, and a copper reducing agent under acidic conditions, while DHSCH was a more potent copper reducing agent under neutral conditions. In the next step, both DHS and DHSCH were tested for metal-based Fenton chemistry in vitro using HPLC with coulometric detection. Neither of these compounds were able to block the iron-based Fenton reaction and, in addition, they mostly intensified hydroxyl radical production. In the copper-based Fenton reaction, the effect of DHSCH was again prooxidant or neutral, while the effect of DHS was profoundly condition-dependent. DHS was even able to attenuate the reaction under some conditions. Interestingly, both compounds were strongly protective against the copper-triggered lysis of red blood cells, with DHSCH being more potent. The results from this study indicated that, notwithstanding the prooxidative effects of both dehydroflavonolignans, their in vivo effect could be protective.

Effect of Small Molecule on ex vivo Expansion of Cord Blood Hematopoietic Stem Cells: A Concise Review

Hematopoietic stem cells (HSCs) are a group of cells being produced during embryogenesis to preserve the blood system. They might also be differentiated to non-hematopoietic cells, including neural, cardiac and myogenic cells. Therefore, they have vast applications in the treatment of human disorders. Considering the restricted quantities of HSCs in the umbilical cord blood, inadequate mobilization of bone marrow stem cells, and absence of ethnic dissimilarity, ex vivo expansion of these HSCs is an applicable method for obtaining adequate amounts of HSCs. Several molecules such as NR-101, zVADfmk, zLLYfmk, Nicotinamide, Resveratrol, the Copper chelator TEPA, dmPGE2, Garcinol, and serotonin have been used in combination of cytokines to expand HSCs ex vivo. The most promising results have been obtained from cocktails that influence multipotency and self-renewal features from different pathways. In the current manuscript, we provide a concise summary of the effects of diverse small molecules on expansion of cord blood HSCs.

DIPG-83. USING COPPER CHELATING AGENTS TO TARGET RECEPTOR TYROSINE KINASE SIGNALLING IN DIFFUSE INTRINSIC PONTINE GLIOMA (DIPG)

DIPG is a universally fatal pediatric brain cancer. Receptor tyrosine kinase (RTK) pathway alterations are among the defining characteristics in many patients. Copper is a transition metal essential for cellular signaling, known to impact PI3K/AKT and MAPK/ERK pathways. Copper chelating agents are clinically approved for use in children with Wilson’s Disease, documented to reduce brain copper levels and are cited as potential cancer therapeutics. Due to copper’s wide cellular integration, we propose that targeting copper in DIPG through use of copper chelators is a viable therapeutic strategy and are strong candidates for combination therapy. Cytotoxicity assays performed in a panel of DIPG cell lines using copper chelator tetraethylenepentamine (TEPA) demonstrated a millimolar range of efficacy. To identify copper integrated pathways, western blots were performed on DIPG cell lines dosed with sub-lethal copper concentrations, which increased phosphorylated expression of AKT, ERK1/2, ERK5 and STAT3. Conversely, western blots performed after TEPA treatment demonstrated reduced phosphorylated expression of all these proteins compared to controls. Western blots investigating TEPA in combination with Everolimus and Trametinib demonstrated synergistic targeting of these proteins. Our results indicate that adding copper in the culture media initiated two RTK-mediated downstream signal transductions, including AKT and ERK and additionally STAT signaling. The use of copper chelator TEPA affected copper homeostasis and reduced DIPG cell proliferation. Our study proposes copper plays an important role in RTK-mediated signaling promoting DIPG proliferation. This implies that reducing copper with clinically available chelation agents can represent a potential anti-cancer treatment for DIPG.

Interaction of 2,6,7-Trihydroxy-Xanthene-3-Ones with Iron and Copper, and Biological Effect of the Most Active Derivative on Breast Cancer Cells and Erythrocytes

Metal chelators can be potentially employed in the treatment of various diseases, ranging from metal overload to neoplastic conditions. Some xanthene derivatives were previously reported to complex metals. Thus, in a search for a novel iron or copper chelator, a series of 9-(substituted phenyl)-2,6,7-trihydroxy-xanthene-3-ones was tested using a competitive spectrophotometric approach. The most promising compound was evaluated in biological models (breast adenocarcinoma cell lines and erythrocytes). In general, substitution of the benzene ring in position 9 had a relatively low effect on the chelation. Only the trifluoromethyl substitution resulted in stronger chelation, probably via a positive effect on solvation. All compounds chelated iron, but their copper-chelating effect was only minimal, since it was no longer observed under highly competitive conditions. Interestingly, all compounds reduced both iron and copper. Additional experiments showed that the trifluoromethyl derivative protected erythrocytes and even cancer cells against excess copper. In summary, the tested compounds are iron chelators, which are also capable of reducing iron/copper, but the copper-reducing effect is not associated with increased copper toxicity.