The curcumin analogue PAC has potent anti-anaplastic thyroid cancer effects.

Anaplastic thyroid carcinoma (ATC) is the rarest type of thyroid cancer, but is the common cause of death from these tumors. The aggressive behavior of ATC makes it resistant to the conventional therapeutic approaches. Thus, the present study was designed to evaluate the anti-ATC efficacy of the piperidone analogue of curcumin (PAC). We have shown that PAC induces apoptosis in thyroid cancer cells in a time-dependent fashion through the mitochondrial pathway. Immunoblotting analysis revealed that PAC suppressed the epithelial-to-mesenchymal transition (EMT) process in ATC cells by upregulating the epithelial marker E-cadherin and reducing the level of the mesenchymal markers N-cadherin, Snail, and Twist1. This anti-EMT effect was confirmed by showing PAC-dependent inhibition of the proliferation and migration abilities of ATC cells. Furthermore, PAC inhibited the AKT/mTOR pathway in ATC cells. Indeed, PAC downregulated mTOR and its downstream effectors p70S6K and 4E-BP1 more efficiently than the well-known mTOR inhibitor rapamycin. In addition to the promising in vitro anticancer efficacy, PAC significantly suppressed the growth of humanized thyroid tumor xenografts in mice. Together, these findings indicate that PAC could be considered as promising therapeutic agent for anaplastic thyroid carcinomas.

Synthesis, Radiolabeling, and Preclinical Evaluation of 68Ga/177Lu-Labeled Leuprolide Peptide Analog for the Detection of Breast Cancer.

Objectives: The expansion of novel and potent tumor receptor binding peptides is a promising approach for the precise targeting of various cancer. Leuprolide is a 9-residue peptide analog of gonadotropin-releasing hormone and is extensively used in the treatment of sex hormone-dependent tumors, including prostate, breast, and ovarian cancer. This preclinical study was undertaken to prepare a new radiolabeled leuprolide peptide for the detection of breast carcinoma. Methods: A 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-coupled 9-amino acid leuprolide peptide was synthesized after typical 9-fluorenylmethyl-oxycarbonyl-based solid-phase peptide synthesis and radiolabeled with both 68Ga and 177Lu radionuclides for theranostic use. The systemic pharmacokinetics was done in healthy balb/c mice. The in vitro tumor cell binding affinity was determined on MCF7, T47D, and MDA-MB-231 breast cancer cell lines. In vivo tumor targeting and micro positron-emission tomography imaging was performed on nude mice with MCF7 breast tumor xenografts. Results: The leuprolide peptide was conveniently synthesized by solid-phase synthesis strategy and its identity and purity were validated by mass spectrometry and high-performance liquid chromatography. The peptide radiolabeled efficiently (˃94%) with both diagnostic (68Ga) and therapeutic (177Lu) radionuclides and displayed nanomolar binding potency to all three tested MCF7, T47D, and MDA-MB-231 cell lines. Fast and favorable pharmacokinetics was observed for 68Ga/177Lu-leuprolide in healthy Balb/c mice. In nude mice, 68Ga-leuprolide peptide exhibited rapid clearance from the blood circulation with low to moderate (up to 5% ID/g) uptake/retention by the major body organs. The accumulation in the estrogen receptor-positive MCF7 tumor was 2.24% ± 0.62% ID/g at 45 min p.i, with good tumor to blood and muscle uptake ratios. The radiolabeled peptide was excreted primarily through the renal pathway. Conclusion: The encouraging results of this initial study demonstrate that additional testing of this leuprolide peptide seems to be indicated because of its convincing potential to be a new agent for the management of breast carcinoma.

ß-Catenin Attenuation Inhibits Tumor Growth and Promotes Differentiation in a BRAFV600E-Driven Thyroid Cancer Animal Model.

BRAFV600E mutation is the most frequent genetic alteration in papillary thyroid cancer (PTC). ß-Catenin (Ctnnb1) is a key downstream component of canonical Wnt signaling pathway and is frequently overexpressed in PTC. BRAF V600E-driven tumors have been speculated to rely on Wnt/ß-catenin signaling to sustain its growth, although many details remain to be elucidated. In this study, we investigated the role of ß-catenin in BrafV600E -driven thyroid cancer in a transgenic mouse model. In Braf V600E mice with wild-type (WT) Ctnnb1 (BVE-Ctnnb1WT or BVE), overexpression of ß-catenin was observed in thyroid tumors. In Braf V600E mice with Ctnnb1 knockout (BVE-Ctnnb1null), thyroid tumor growth was slowed with significant reduction in papillary architecture. This was associated with increased expression of genes involved in thyroid hormone synthesis, elevated 124iodine uptake, and serum T4. The survival of BVE-Ctnnb1null mice was increased by more than 50% during 14-month observation. Mechanistically, downregulation of MAPK, PI3K/Akt, and TGFß pathways and loss of epithelial-mesenchymal transition (EMT) were demonstrated in the BVE-Ctnnb1null tumors. Treatment with dual ß-catenin/KDM4A inhibitor PKF118-310 dramatically improved the sensitivity of BVE-Ctnnb1WT tumor cells to BRAFV600E inhibitor PLX4720, resulting in significant growth arrest and apoptosis in vitro, and tumor regression and differentiation in vivo These findings indicate that ß-catenin signaling plays an important role in thyroid cancer growth and resistance to BRAFV600E inhibitors. Simultaneously targeting both Wnt/ß-catenin and MAPK signaling pathways may achieve better therapeutic outcome in BRAFV600E inhibitor-resistant and/or radioiodine-refractory thyroid cancer.

The curcumin analog (PAC) suppressed cell survival and induced apoptosis and autophagy in oral cancer cells.

PAC (3,5-Bis (4-hydroxy-3-methoxybenzylidene)-N-methyl-4-piperidone), a novel bioactive curcumin analog, has been reported to have anticancer properties against various tumors. However, the anti-cancer effects of PAC on oral cavity squamous cell carcinoma were not studied yet. Our aim is to investigate the anti-oral cancer properties of PAC in vitro, and determine the molecular mechanisms underlying these effects. Viability assays including MTT and LDH were conducted to measure cell proliferation. Flow cytometry-based cytotoxicity assay was performed to detect autophagic cell death and oxidative stress markers. Western blotting was used for measuring protein expression/activation in apoptotic, autophagic and pro-carcinogenic cellular signaling pathways. We demonstrated that PAC preferentially and, in a dose, -dependent way kills oral cancer cells, but was not toxic to normal human gingival cells. PAC destabilizes cell-cycle distributions, inhibits the expression of oncogenes (cyclin D1) and that of cyclin-dependent kinase inhibitor (p21WAF1) is upregulated, increases the expression of p53 gene, and inhibits epithelial-mesenchymal transition markers in oral cancer cells. The PAC effect involve various signaling pathways including NF-κB, MAPK, Wnt, caspase-3/9 and PARP1. Finally, PAC demonstrated ability to induce autophagy, decrease production of reactive oxygen species, increase intracellular glutathione (GSH) activity, and reduce mitochondrial membrane potential in oral cancer cells. In conclusion, PAC inhibits the proliferation and increases the apoptosis and autophagy and oxidative stress of oral cancer cells. These effects involve ERK1/2, p38/JNK, NF-κB and Wnt cellular signaling pathways. Overall, our study suggests the potential use of PAC to treat oral cancer.

PAC down-regulates estrogen receptor alpha and suppresses epithelial-to-mesenchymal transition in breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is an aggressive histological subtype with limited treatment options and very poor prognosis following progression after standard chemotherapeutic regimens. Therefore, novel molecules and therapeutic options are urgently needed for this category of patients. Recently, we have identified PAC as a curcumin analogue with potent anti-cancer features. METHODS: HPLC was used to evaluate the stability of PAC and curcumin in PBS and also in circulating blood. Cytotoxicity/apoptosis was assessed in different breast cancer cell lines using propidium iodide/annexinV associated with flow cytometry. Furthermore, immunoblotting analysis determined the effects of PAC on different oncogenic proteins and pathways. Additionally, the real time xCELLigence RTCA technology was applied to investigate the effect of PAC on the cellular proliferation, migration and invasion capacities. RESULTS: PAC is more stable than curcumin in PBS and in circulating blood. Furthermore, we have shown differential sensitivity of estrogen receptor-alfa positive (ERα(+)) and estrogen receptor alfa negative (ERα(-)) breast cancer cells to PAC, which down-regulated ERα in both cell types. This led to complete disappearance of ERα in ERα(-) cells, which express very low level of this receptor. Interestingly, specific down-regulation of ERα in receptor positive cells increased the apoptotic response of these cells to PAC, confirming that ERα inhibits PAC-dependent induction of apoptosis, which could be mediated through ERα down-regulation. Additionally, PAC inhibited the proliferation and suppressed the epithelial-to-mesenchymal transition process in breast cancer cells, with higher efficiency on the TNBC subtype. This effect was also observed in vivo on tumor xenografts. Additionally, PAC suppressed the expression/secretion of 2 important cytokines IL-6 and MCP-1, and consequently inhibited the paracrine procarcinogenic effects of breast cancer cells on breast stromal fibroblasts. CONCLUSION: These results indicate that PAC could be considered as important candidate for future therapeutic options against the devastating TNBC subtype.

Preparation and evaluation of the tumor-specific antigen-derived synthetic mucin 1 peptide: A potential candidate for the targeting of breast carcinoma.

PURPOSE: The goal of this study was to prepare a synthetic peptide derived from breast tumor associated antigen and to evaluate its potential as a breast cancer imaging agent. METHODS: A mucin 1 derived peptide was synthesized by solid-phase peptide synthesis and examined for its radiochemical and metabolic stability. The tumor cell binding affinity of (99m)Tc-MUC1 peptide was investigated on MUC1-positive T47D and MCF7 breast cancer cell lines. In vivo biodistribution was studied in normal Balb/c mice and in vivo tumor targeting and imaging in MCF7 and T47D tumor-bearing nude mice. RESULTS: The synthesized MUC1-derived peptide displayed high radiochemical and metabolic stability. In vitro tumor cell-binding on T47D and MCF7 cell lines demonstrated high affinity of (99m)Tc-MUC1 peptide towards human breast cancer cells (binding affinities in nanomolar range). Pharmacokinetic studies performed on Balb/c mice are characterized by an efficient clearance from the blood and excretion predominantly through the urinary system. In vivo tumor uptake in nude mice with MCF7 tumor xenografts was 2.77±0.63% ID/g as early as 1h p.i. whereas in nude mice with T47D human ductal breast epithelial cancer cells, the accumulation in the tumor was found to be 2.65±0.54% ID/g at 1h p.i. Also tumor lesion was detectable in γ-camera imaging. The tumor uptake values were always higher than the blood and muscle uptake, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. A low to moderate (<5% ID/g) accumulation and retention of (99m)Tc-MUC1 was found in the major organs (i.e., lungs, stomach, liver, intestines, kidneys, etc.) in both normal and tumor-bearing mice. CONCLUSION: This study suggests that (99m)Tc-MUC1 tumor-antigen peptide may be a potential candidate for the targeted imaging of MUC1-positive human tumors and warrants further investigation.

Specific targeting and noninvasive imaging of breast cancer stem cells using single-walled carbon nanotubes as novel multimodality nanoprobes.

BACKGROUND: The limitation of current breast cancer treatments was elucidated by the presence of cancer stem cells (CSCs) that play essential role in cancer initiation, progression, resistance, recurrence and metastasis. Materials & methods: Biocompatible multimodality single-walled carbon nanotube (SWCNT) nanoprobes were developed. The biodistribution and preferential homing of CD44 antibody-conjugated SWCNTs toward the tumor site were monitored using MRI, single-photon emission computed tomography and near-infrared fluorescence imaging noninvasive imaging modalities. RESULTS: Quantification of SWCNTs by sensitively measuring iron content in sorted CSC populations using inductively coupled plasma-mass spectrometry confirmed the enhanced selective targeting of anti-CD44 SWCNT and immunohistochemistry analyses revealed enhanced colocalization with areas rich in CD44 receptors. DISCUSSION & CONCLUSION: This preclinical study provided encouraging results for efficient targeting of breast CSCs and perspectives for further clinical studies to confirm the efficacy and safety of the designed nanocarriers.

PAC exhibits potent anti-colon cancer properties through targeting cyclin D1 and suppressing epithelial-to-mesenchymal transition.

Colorectal cancer (CRC) is a major cause of cancer morbidity and mortality worldwide. Although response rates and overall survival have been improved in recent years, resistance to multiple drug combinations is inevitable. Therefore, the development of more efficient drugs, with fewer side effects is urgently needed. To this end, we have investigated in the present report the effect of PAC, a novel cucumin analogue, on CRC cells both in vitro and in vivo. We have shown that PAC induces apoptosis, mainly via the internal mitochondrial route, and inhibits cell proliferation through delaying the cell cycle at G2/M phase. Interestingly, the pro-apoptotic effect was mediated through STAT3-dependent down-regulation of cyclin D1 and its downstream target survivin. Indeed, change in the expression level of cyclin D1 modulated the expression of survivin and the response of CRC cells to PAC. Furthermore, using the ChIP assay, we have shown PAC-dependent reduction in the binding of STAT3 to the cyclin D1 promoter in vivo. Additionally, PAC suppressed the epithelial-to-mesenchymal process through down-regulating the mesenchymal markers (N-cadherin, vimentin and Twist1) and inhibiting the invasion/migration abilities of the CRC cells via repressing the pro-migration/invasion protein kinases AKT and ERK1/2. In addition, PAC inhibited tumor growth and repressed the JAK2/STAT3, AKT/mTOR and MEK/ERK pathways as well as their common downstream effectors cyclin D1 and survivin in humanized CRC xenografts. Collectively, these results indicate that PAC has potent anti-CRC effects, and therefore could constitute an effective alternative chemotherapeutic agent, which may consolidate the adjuvant treatment of colon cancer.

Sodium-22-radiolabeled silica nanoparticles as new radiotracer for biomedical applications: in vivo positron emission tomography imaging, biodistribution, and biocompatibility.

Despite their advantageous chemical properties for nuclear imaging, radioactive sodium-22 ((22)Na) tracers have been excluded for biomedical applications because of their extremely long lifetime. In the current study, we proposed, for the first time, the use of (22)Na radiotracers for pre-clinical applications by efficiently loading with silica nanoparticles (SiNPs) and thus offering a new life for this radiotracer. Crown-ether-conjugated SiNPs (300 nm; -0.18±0.1 mV) were successfully loaded with (22)Na with a loading efficacy of 98.1%±1.4%. Noninvasive positron emission tomography imaging revealed a transient accumulation of (22)Na-loaded SiNPs in the liver and to a lower extent in the spleen, kidneys, and lung. However, the signal gradually decreased in a time-dependent manner to become not detectable starting from 2 weeks postinjection. These observations were confirmed ex vivo by quantifying (22)Na radioactivity using γ-counter and silicon content using inductively coupled plasma-mass spectrometry in the blood and the different organs of interest. Quantification of Si content in the urine and feces revealed that SiNPs accumulated in the organs were cleared from the body within a period of 2 weeks and completely in 1 month. Biocompatibility evaluations performed during the 1-month follow-up study to assess the possibility of synthesized nanocarriers to induce oxidative stress or DNA damage confirmed their safety for pre-clinical applications. (22)Na-loaded nanocarriers can thus provide an innovative diagnostic agent allowing ultra-sensitive positron emission tomography imaging. On the other hand, with its long lifetime, onsite generators or cyclotrons will not be required as (22)Na can be easily stored in the nuclear medicine department and be used on-demand.

Camel urine components display anti-cancer properties in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: While camel urine (CU) is widely used in the Arabian Peninsula to treat various diseases, including cancer, its exact mechanism of action is still not defined. The objective of the present study is to investigate whether camel urine has anti-cancer effect on human cells in vitro. MATERIALS AND METHODS: The annexinV/PI assay was used to assess apoptosis, and immunoblotting analysis determined the effect of CU on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilized to investigate cytotoxicity and the effect on the cell cycle as well as the production of cytokines, respectively. RESULTS: Camel urine showed cytotoxicity against various, but not all, human cancer cell lines, with only marginal effect on non-tumorigenic epithelial and normal fibroblast cells epithelial and fibroblast cells. Interestingly, 216 mg/ml of lyophilized CU inhibited cell proliferation and triggered more than 80% of apoptosis in different cancer cells, including breast carcinomas and medulloblastomas. Apoptosis was induced in these cells through the intrinsic pathway via Bcl-2 decrease. Furthermore, CU down-regulated the cancer-promoting proteins survivin, ß-catenin and cyclin D1 and increased the level of the cyclin-dependent kinase inhibitor p21. In addition, we have shown that CU has no cytotoxic effect against peripheral blood mononuclear cells and has strong immuno-inducer activity through inducing IFN-γ and inhibiting the Th2 cytokines IL-4, IL-6 and IL-10. CONCLUSIONS: CU has specific and efficient anti-cancer and potent immune-modulator properties in vitro.

PAC, a novel curcumin analogue, has anti-breast cancer properties with higher efficiency on ER-negative cells.

We have investigated here the anti-breast cancer properties of two novel curcumin analogues, EAC and PAC. Apoptosis was assessed by the annexin V/propidium iodide (PI) assay on different breast cancer and normal cells. Immunoblotting analysis determined the effects of these agents on different apoptotic and oncogenic proteins. Furthermore, flow cytometry and Elispot were utilised to investigate the effects on the cell cycle and the production of cytokines, respectively. Breast cancer tumour xenografts were developed in nude mice. Finally, (18)F-radiolabeled PAC and curcumin were produced to study their bioavailability and tissue biodistribution in mice. PAC is five times more efficient than curcumin and EAC in inducing apoptosis, mainly via the internal mitochondrial route. This effect was 10-fold higher against ER-negative as compared to ER-positive cells, and ectopic expression of ERα rendered ER-negative breast cancer cells more resistant to PAC. In addition, PAC delayed the cell cycle at G2/M phase with a stronger effect on ER-negative cells. Moreover, PAC exhibited strong capacity as an immuno-inducer through reducing the secretion of the two major Th2 cytokines IL-4 and IL-10. Importantly, PAC significantly reduced tumour size, and triggered apoptosis in vivo. Furthermore, PAC inhibited survivin, NF-kB and its downstream effectors cyclin D1 and Bcl-2, and strongly up-regulated p21(WAF1) both in vitro and in tumours. Besides, PAC exhibited higher stability in blood and greater biodistribution and bioavailability than curcumin in mice. These results indicate that PAC could constitute a powerful, yet not toxic, new chemotherapeutic agent against ER-negative breast tumours.

Synthesis and evaluation of a technetium-99m labeled cytotoxic bombesin peptide conjugate for targeting bombesin receptor-expressing tumors.

Conjugation of the cytotoxic drugs to receptor-binding peptides is an attractive approach for the targeted delivery of cytotoxic peptide conjugates to tumor cells. In an attempt to develop an efficient peptide-based radiopharmaceutical for targeting bombesin (BN) receptor-expressing tumors (i.e., breast and prostate), we have prepared by solid-phase peptide synthesis, a novel BN analog derived from the universal sequence of BN and conjugated to a widely characterized antineoplastic agent, methotrexate (MTX). MTX-BN, after radiolabeling with (99m)Tc via stannous-tartrate exchange, showed a good stability against cysteine and histidine transchelation as well as a high in vitro metabolic stability in human plasma. In vitro cell-binding and internalization on MDA-MB-231, MCF-7, T47-D breast cancer and PC-3 prostate cancer cell lines demonstrated high affinity and specificity of (99m)Tc-MTX-BN towards both human breast and prostate cancer cells (binding affinities in nanomolar range). In addition, the radioconjugate displayed a significant internalization (values ranged between 19-35%) into the tumor cells. In vivo biodistribution and clearance kinetics in Balb/c mice are characterized by an efficient clearance from the blood and excretion mainly through the renal-urinary pathway with some elimination via the hepatobiliary system. In vivo tumor uptake in nude mice bearing MDA-MB-231 cells was 2.70+/-0.44% ID/g at 1 h, whereas in nude mice with human epidermoid KB cells the accumulation in the tumor was found to be 1.48+/-0.31% ID/g at 1 h post injection. The tumor uptake was always higher than in the blood and muscle, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. The accumulation/retention in the major organs (i.e., lungs, stomach, liver, intestines, etc.) was low to moderate (<6% ID/g) in both healthy and tumor-bearing mice. However, the uptake/retention in the kidneys was rather high (up to 11.05+/-1.80% ID/g), which is of a concern, particularly for radionuclide therapy. This initial study towards the development of a novel cytotoxic BN conjugate suggest that the combination of favorable in vitro and in vivo properties may render (99m)Tc-MTX-BN a potential candidate for the targeted imaging and eventually for radionuclide therapy (when labeled with an appropriate radionuclide) of BN receptor-positive tumors and deserves further evaluation.

Radioiodinated naphthylalanine derivatives targeting pancreatic beta cells in normal and nonobese diabetic mice.

An imaging method capable of using a signal from pancreatic beta cells to determine their mass would be of immense value in monitoring the progression of diabetes as well as response to treatment. Somatostatin receptors (SSTRs) are expressed on beta cells and are a potential target for imaging. The main objective of this study was to investigate whether pancreatic beta cells are a target for radiolabeled naphthylalanine derivatives. The molecules were subjected to in vitro and ex vivo evaluations. Pancreatic uptake of radioactivity was lower in nonobese diabetic (NOD) mice than normal mice at all time points investigated (P < .05) and correlated with the number of islets in tissue sections of both control and NOD mice. Immunohistochemical and confocal fluorescent microscopic studies showed colocalization of insulin and the conjugate radioligand in the pancreas. The results demonstrated that pancreatic uptake is receptor-mediated, and that beta cells are the primary target.