Reduction in Copper Uptake and Inhibition of Prostate Cancer Cell Proliferation by Novel Steroid-based Compounds.

BACKGROUND/AIM: Knockdown of human copper transporter 1 has been associated with reduction in copper uptake and suppression of prostate cancer cell proliferation and tumor growth. This study evaluated the effects of steroid-based compounds on copper uptake and proliferation of prostate cancer cells based on their anticancer activity and previous docking analysis of steroid-based copper transporter 1 inhibitors. MATERIALS AND METHODS: We synthesized several new steroid-based compounds and used 64Cu uptake assay and copper quantification assay with inductively coupled plasma mass spectrometry to study their effects on the cellular copper uptake by prostate cancer cells. Additionally, we used CCK-8 cell proliferation assay to study their effects on the proliferation of prostate cancer cells. RESULTS: Significant reduction in cellular copper uptake was observed in the prostate cancer cells treated with these new steroid-based compounds. Moreover, proliferation of prostate cancer cells was suppressed by treatment with the steroid-based compound 6, which had the strongest copper uptake inhibition activity. CONCLUSION: Reduction in copper uptake and inhibition of cell proliferation were demonstrated in prostate cancer cells treated with the new steroid-based compounds synthesized in this study. Steroid-based copper transporter 1 inhibitors may become novel anticancer drugs for targeted anti-copper therapy of prostate cancer and other copper hypermetabolic cancers.

Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance.

BACKGROUND: Radiation therapy remains an important treatment modality in cancer therapy, however, resistance is a major problem for treatment failure. Elevated expression of glutathione is known to associate with radiation resistance. We used glutathione overexpressing small cell lung cancer cell lines, SR3A-13 and SR3A-14, established by transfection with γ-glutamylcysteine synthetase (γ-GCS) cDNA, as a model for investigating strategies of overcoming radiation resistance. These radiation-resistant cells exhibit upregulated human copper transporter 1 (hCtr1), which also transports cisplatin. This study was initiated to investigate the effect and the underlying mechanism of iron-platinum nanoparticles (FePt NPs) on radiation sensitization in cancer cells. MATERIALS AND METHODS: Uptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry and transmission electron microscopy. Effects of the combination of FePt NPs and ionizing radiation were investigated by colony formation assay and animal experiment. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a fluorescence-activated-cell-sorting caliber flow cytometer. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahorse XF24 cell energy metabolism analyzer. RESULTS: These hCtr1-overexpressing cells exhibited elevated resistance to IR and the resistance could be overcome by FePt NPs via enhanced uptake of FePt NPs. Overexpression of hCtr1 was responsible for the increased uptake/transport of FePt NPs as demonstrated by using hCtr1-transfected parental SR3A (SR3A-hCtr1-WT) cells. Increased ROS and drastic mitochondrial damages with substantial reduction of oxygen consumption rate were observed in FePt NPs and IR-treated cells, indicating that structural and functional insults of mitochondria are the lethal mechanism of FePt NPs. Furthermore, FePt NPs also increased the efficacy of radiotherapy in mice bearing SR3A-hCtr1-WT-xenograft tumors. CONCLUSION: These results suggest that FePt NPs can potentially be a novel strategy to improve radiotherapeutic efficacy in hCtr1-overexpressing cancer cells via enhanced uptake and mitochondria targeting.

Preclinical PET imaging study of lung cancer with 64CuCl2.

OBJECTIVE: Human copper transporter 1 (CTR1) has been proven to be overexpressed in many types of cancer cells, and copper (II)-64 chloride (64CuCl2) has been used as an effective tracer for positron emission tomography (PET) imaging in tumor-bearing animal models. Thus, this study aimed to investigate the potential application of 64CuCl2 in PET imaging of lung cancer through targeting CTR1. METHODS: The expression of CTR1 in a series of lung cancer cell lines was identified by quantitative real-time polymerase chain reaction (Q-PCR), western blot, enzyme-linked immunosorbnent assay (ELISA), and immunofluorescent staining. Then in vitro cell uptake assay of 64CuCl2 was investigated in human lung cancer cell lines with different levels of CTR1 expression. Small animal PET imaging and quantitative analysis were performed in human lung cancer tumor-bearing mice after intravenous injection of 64CuCl2, respectively. RESULTS: The CTR1 expression in multiple human lung cancer cells was identified and confirmed, and H1299 cell lines with high CTR1 expression, H460 with moderate CTR1, and H1703 with low CTR1 were selected for further experiments. In vitro cellular uptake assay displayed that the 64CuCl2 uptake by these three kinds of cells was positively correlated with their CTR1 expressed levels. The blocking experiments testified the specificity of 64CuCl2 to target CTR1. Moreover, small animal PET imaging and quantitative results showed that 64CuCl2 accumulation in H1299, H460, and H1703 tumor-bearing mice were consistent with CTR1 levels and cell uptake experiments. CONCLUSIONS: The expression of CTR1 in human lung cancer xenograft model could be successfully visualized by 64CuCl2 PET examination. With the expected growth of PET/CT examination to be an essential strategy in clinical lung cancer management, 64CuCl2 has the potential to be a promising PET imaging agent of lung cancer.

A new metal affinity NCTR25 tag as a better alternative to the His-tag for the expression of recombinant fused proteins.

His-tagging is commonly used in fusion protein production, but the His-tag is usually prohibited in medicinal proteins and must be removed. A fragment (NCTR25-tag) truncated from the N-terminus of human copper transporter 1 was tested for feasibility as a replacement for the His-tag in fusion proteins. The NCTR25-tag and His-tag were separately fused to the transthyretin (TTR) protein, and the expression, affinity purification, refolding and stability of the two kinds of fusions were compared. NCTR25 fusion produced a 63% higher yield of the recombinant protein, which was purified by metal affinity chromatography with an efficiency similar to that of His-tagged protein. NCTR25-tag fusion had much less impact on the foldability, kinetic and thermodynamic stability of tetrameric TTR than His-tag fusion. When the tags were individually fused to enhanced green fluorescent protein (EGFP), NCTR25 fusion yielded 29-128% more product than His-EGFP. NCTR25-EGFP could be purified by metal affinity chromatography and showed better foldability than His-EGFP. Furthermore, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) fusion with the third disulfide loop of TGF-α (TGF3L-TRAIL) fused with the NCTR25-tag retained the stability and superactivity of His-TGF3L-TRAIL. Therefore, the native tag NCTR25-tag is a feasible alternative to the His-tag in medicinal recombinant proteins.

A Dose Escalation Study of Trientine Plus Carboplatin and Pegylated Liposomal Doxorubicin in Women With a First Relapse of Epithelial Ovarian, Tubal, and Peritoneal Cancer Within 12 Months After Platinum-Based Chemotherapy.

Background: Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer-related deaths worldwide. Preclinical studies found that copper-lowering agents could re-sensitize platinum-resistant cancer cells by enhancing the human copper transporter 1 (hCtr1)-mediated uptake of platinum. In the clinic, re-sensitization of platinum-resistance in relapsed EOC has been discovered by the application of trientine plus platinum (NCT01178112). However, no pharmacokinetic data of trientine has been reported in cancer patients. Purpose: Our study aimed to explore the safety and activity of trientine combined with carboplatin and pegylated liposomal doxorubicin (PLD) in patients with EOC, tubal, and peritoneal cancer who experienced disease progression during platinum-based chemotherapy or showed relapse <12 months after completing first-line chemotherapy. Also, we aimed to demonstrate pharmacokinetic parameters and to discover potential biomarkers in our EOC patients. Methods: In this dose escalation study, 18 Asian patients in six dosing cohorts received fixed doses of carboplatin (AUC 4) and PLD (LipoDox®, TTY Biopharm Co. Ltd., Taipei, Taiwan) (40 mg/m2, day 1 per 4-week cycle), and escalated daily trientine doses (range: 300-1800 mg; initiated 7 days before the 1st combination cycle) according to a 3 + 3 design. Results: No dose-limiting toxicity or treatment-related death was observed. Four patients (22.2%) developed grade 3 drug-related adverse events (AEs), whereas no grade 4 AEs were encountered. Anemia and grade 2 dizziness were the most common hematological toxicity and neurotoxicity, respectively. In a pharmacokinetics comparison with healthy volunteers in the literature, our patients achieved greater absorption after oral trientinem, and more rapid elimination of triethylenetetramine dihydrochloride at high doses. The clinical benefit rate was 33.3 and 50.0% in the platinum-resistant and the partially platinum-sensitive group, respectively. A high baseline serum iron level and low serum copper level might help differentiate subgroups of patients with different clinical responses. Nevertheless, no associations of the clinical response with the levels of serum hCtr1, ceruloplasmin, or copper were observed. Conclusion: Combination therapy with carboplatin, trientine, and PLD was well-tolerated and safe. Our results encourage the development of a future phase II trial. Clinical trial registration: ClinicalTrials.gov # NCT03480750.

Rapid diagnosis of cisplatin-sensitive and resistant cervical squamous cell carcinomas by reverse transcription loop-mediated isothermal amplification.

Cisplatin is a widely used platinum anticancer drug in cervical cancer chemotherapy; however, its use is limited by the development of drug resistance, which is often rationalized via effects on cellular uptake. Human copper transporter 1 (hCTR1) was proven as crucial regulator for cellular platinum uptake in cancer cells and improving effect of cisplatin treatment. Thus, methods to detect expression of hCTR1 in patients in biopsy specimens with certain rapidity and accuracy are essential to evaluate platinum drug sensitivity and perform further individualized treatment. In this study, a total of 46 normal cervical and primary cervical squamous cell carcinoma tissues were collected and tested by reverse transcription loop-mediated isothermal amplification (RT-LAMP) analysis targeted hCTR1 mRNA. Sensitivity and specificity of RT-LAMP results were calculated and compared to immunohistochemical (IHC) examination. Overexpression of hCTR1 RNA was detected in 12 samples using RT-LAMP within 45 minutes reaction, which indicated positivity with cisplatin resistance. This new technique is suggested to be alternative for rapid diagnosis of cisplatin sensitive patients whose hCTR1 is prevalent.

Reduced 64Cu uptake and tumor growth inhibition by knockdown of human copper transporter 1 in xenograft mouse model of prostate cancer.

UNLABELLED: Copper is an element required for cell proliferation and angiogenesis. Human prostate cancer xenografts with increased (64)Cu radioactivity were visualized previously by PET using (64)CuCl2 as a radiotracer ((64)CuCl2 PET). This study aimed to determine whether the increased tumor (64)Cu radioactivity was due to increased cellular uptake of (64)Cu mediated by human copper transporter 1 (hCtr1) or simply due to nonspecific binding of ionic (64)CuCl2 to tumor tissue. In addition, the functional role of hCtr1 in proliferation of prostate cancer cells and tumor growth was also assessed. METHODS: A lentiviral vector encoding short-hairpin RNA specific for hCtr1 (Lenti-hCtr1-shRNA) was constructed for RNA interference-mediated knockdown of hCtr1 expression in prostate cancer cells. The degree of hCtr1 knockdown was determined by Western blot, and the effect of hCtr1 knockdown on copper uptake and proliferation were examined in vitro by cellular (64)Cu uptake and cell proliferation assays. The effects of hCtr1 knockdown on tumor uptake of (64)Cu were determined by PET quantification and tissue radioactivity assay. The effects of hCtr1 knockdown on tumor growth were assessed by PET/CT and tumor size measurement with a caliper. RESULTS: RNA interference-mediated knockdown of hCtr1 was associated with the reduced cellular uptake of (64)Cu and the suppression of prostate cancer cell proliferation in vitro. At 24 h after intravenous injection of the tracer (64)CuCl2, the (64)Cu uptake by the tumors with knockdown of hCtr1 (4.02 ± 0.31 percentage injected dose per gram [%ID/g] in Lenti-hCtr1-shRNA-PC-3 and 2.30 ± 0.59 %ID/g in Lenti-hCtr1-shRNA-DU-145) was significantly lower than the (64)Cu uptake by the control tumors without knockdown of hCtr1 (7.21 ± 1.48 %ID/g in Lenti-SCR-shRNA-PC-3 and 5.57 ± 1.20 %ID/g in Lenti-SCR-shRNA-DU-145, P < 0.001) by PET quantification. Moreover, the volumes of prostate cancer xenograft tumors with knockdown of hCtr1 (179 ± 111 mm(3) for Lenti-hCtr1-shRNA-PC-3 or 39 ± 22 mm(3) for Lenti-hCtr1-shRNA-DU-145) were significantly smaller than those without knockdown of hCtr1 (536 ± 191 mm(3) for Lenti- SCR-shRNA-PC-3 or 208 ± 104 mm(3) for Lenti-SCR-shRNA-DU-145, P < 0.01). CONCLUSION: Overall, data indicated that hCtr1 is a promising theranostic target, which can be further developed for metabolic imaging of prostate cancer using (64)CuCl2 PET/CT and personalized cancer therapy targeting copper metabolism.

Overcoming platinum drug resistance with copper-lowering agents.

Platinum (Pt)-based antitumor agents have been the mainstay of cancer chemotherapy for the last three decades. While multiple mechanisms are responsible for treatment failure, deficiency in drug transport is an important contributor. The human high-affinity copper (Cu) transporter-1 (hCtr1) can also transport Pt-based drugs including cisplatin (cDDP) and carboplatin. Reduced hCtr1 expression frequently occurs in cDDP-resistant cell lines and in cancer in patients who failed chemotherapy with these drugs. We previously demonstrated that Cu chelation induces the expression of transcription factor Sp1 which binds the promoters of Sp1 and hCtr1, thereby, up-regulating their expression, whereas Cu overload shuts down hCtr1 and Sp1 expression by dissociating Sp1 from their promoter promoters. Thus, mammalian Cu homeostasis is transcriptionally regulated within a loop consisting of Sp1, hCtr1, and Cu in a three-way mutually regulated manner. These findings suggest that it is feasible to module cDDP transport capacity through intervention of mammalian Cu homeostasis. Indeed, we found that cDDP resistance can be overcome by Cu-lowering agents through enhanced hCtr1 expression by up-regulation of Sp1 in cultured cells. This discovery provided a mechanistic basis for the ongoing clinical study using Cu chelator to overcome cDDP resistance in ovarian cancer chemotherapy. Preliminary study using copper chelator (trientine) for enhancing the treatment efficacy of carboplatin in 5 ovarian cancer patients showed encouraging results. This short review describes the perspectives of using Cu-lowering agents in overcoming Pt resistance in cancer chemotherapy.