Anti­metastatic activity of aromatic aminomethylidenebisphosphonates in a mouse model of 4T1 cell­derived breast cancer.

Breast cancer is one of the major causes of cancer­related mortality among women worldwide. It metastasizes to distant organs, particularly to bone tissue. Nitrogen­containing bisphosphonates are mainly used as an adjuvant therapy to inhibit skeletal­related events; however, there is increasing evidence to suggest that these compounds also exert antitumor effects. In previous studies, the authors synthesized two novel aminomethylidenebisphosphonates (BPs), namely benzene­1,4­bis[aminomethylidene(bisphosphonic)] acid (WG12399C) and naphthalene­1,5­bis[aminomethylidene(bisphosphonic)] acid (WG12592A). Both BPs exhibited notable antiresorptive activity in a mouse model of osteoporosis. The present study aimed to assess the in vivo anticancer activity of WG12399C and WG12592A in 4T1 breast adenocarcinoma model. WG12399C exerted an anti­metastatic effect by reducing the number of spontaneous lung metastases by ~66% in comparison to the control. In the experimental metastasis model of 4T1­luc2­tdTomato cells, this compound reduced the incidence of tumor metastases in the lungs by approximately half in comparison to the control. Both WG12399C and WG12595A also significantly reduced the size and/or number of bone metastatic foci. Their pro­apoptotic and anti­proliferative activity may, at least in part, explain the observed effects. Incubation with WG12399C induced an almost 6­fold increase in caspase­3 activity in 4T1 cells. Moreover, cells treated with WG12399C or WG12595A exhibited a 2­fold reduction in invasiveness through Matrigel. Furthermore, both the BPs were able to sensitize the 4T1 cells to cytostatics. In summary, the results of the present study indicate that the examined aminomethylidene­BPs may be of particular interest in the context of combined treatment in breast cancer therapy.

Novel Double-Modified Colchicine Derivatives Bearing 1,2,3-Triazole: Design, Synthesis, and Biological Activity Evaluation.

A series of 1,4-disubstituted 1,2,3-triazoles having 10-demethoxy-10-N-methylaminocolchicine core were designed and synthesized via the Cu(I)-catalyzed "click" reaction and screened for their in vitro cytotoxicity against four cancer cell lines (A549, MCF-7, LoVo, LoVo/DX) and one noncancerous cell line (BALB/3T3). Indexes of resistance (RI) and selectivity (SI) were also determined to assess the potential of the analogues to break drug resistance of the LoVo/DX cells and to verify their selectivity toward killing cancer cells over normal cells. The compounds with an ester or amide moiety in the fourth position of 1,2,3-triazole of 10-N-methylaminocolchicine turned out to have the greatest therapeutic potential (low IC50 values and favorable SI values), much better than that of unmodified colchicine or doxorubicin and cisplatin. Thus, they make a valuable clue for the further search for a drug having a colchicine scaffold.

Synthesis and antiproliferative screening of novel doubly modified colchicines containing urea, thiourea and guanidine moieties.

A new series of 10-demethoxy-10-methylaminocolchicines bearing urea, thiourea or aguanidine moieties at position C7 has been designed, synthesized and evaluated for in vitro anticancer activity against different cancer cell lines (A549, MCF-7, LoVo, LoVo/DX). The majority of the new derivatives were active in the nanomolar range and were characterized by lower IC50 values than cisplatin or doxorubicin. Two ureas (4 and 8) and thioureas (19 and 25) were found to be good antiproliferative agents (low IC50 values and high SI) and could prove to be promising candidates for further research in the field of anticancer drugs based on the colchicine skeleton.

Retinol-Binding Protein 4 Accelerates Metastatic Spread and Increases Impairment of Blood Flow in Mouse Mammary Gland Tumors.

Retinol-binding protein 4 (RBP4) is proposed as an adipokine that links obesity and cancer. We analyzed the role of RBP4 in metastasis of breast cancer in patients and in mice bearing metastatic 4T1 and nonmetastatic 67NR mammary gland cancer. We compared the metastatic and angiogenic potential of these cells transduced with Rbp4 (4T1/RBP4 and 67NR/RBP4 cell lines). Higher plasma levels of RBP4 were observed in breast cancer patients with metastatic tumors than in healthy donors and patients with nonmetastatic cancer. Increased levels of RBP4 were observed in plasma, tumor tissue, liver, and abdominal fat. Moreover, the blood vessel network was highly impaired in mice bearing 4T1 as compared to 67NR tumors. RBP4 transductants showed further impairment of blood flow and increased metastatic potential. Exogenous RBP4 increased lung settlement by 67NR and 4T1 cells. In vitro studies showed increased invasive and clonogenic potential of cancer cells treated with or overexpressing RBP4. This effect is not dependent on STAT3 phosphorylation. RBP4 enhances the metastatic potential of breast cancer tumors through a direct effect on cancer cells and through increased endothelial dysfunction and impairment of blood vessels within the tumor.

Calcitriol Analogues Decrease Lung Metastasis but Impair Bone Metabolism in Aged Ovariectomized Mice Bearing 4T1 Mammary Gland Tumours.

Calcitriol and its analogues are considered drugs supporting the anticancer treatment of breast cancer and preventing the osteoporosis that results from the development of cancer or from chemotherapy or hormone therapy. Following the orthotopic implantation of 4T1 mammary carcinoma cells into aged ovariectomized (OVX) mice, we evaluated the effects of calcitriol and its two analogues, PRI-2191 and PRI-2205, on metastatic spread and bone homeostasis. Calcitriol and its analogues temporarily inhibited the formation of metastases in the lungs. Unexpectedly, only mice treated with calcitriol analogues showed a deterioration of bone-related parameters, such as bone column density, marrow column density and the CaPO4 coefficient. These findings correlated with an increased number of active osteoclasts differentiated from bone marrow-derived macrophages in mice treated with the analogues. Interestingly, in the tumours from mice treated with PRI-2191 and PRI-2205, the expression of Tnfsf11 (RANKL) was increased. On the other hand, osteopontin (OPN) levels in plasma and tumour tissue, as well as TRAC5b levels in tumours, were diminished by calcitriol and its analogues. Despite a similar action of both analogues towards bone metabolism, their impact on vitamin D metabolism differed. In particular, PRI-2191 and calcitriol, not PRI-2205 treatment significantly diminished the levels of both 25(OH)D3 and 24,25(OH)2D3. In conclusion, though there is evident antimetastatic activity in old OVX mice, signs of increased bone metabolism and deterioration of bone mineralization during therapy with calcitriol analogues were observed.

Molecular docking studies, biological and toxicity evaluation of dihydroisoquinoline derivatives as potential anticancer agents.

We report a study of a series of isoquinoline derivatives, including their synthesis, in vitro microsomal leucine aminopeptidase (LAP) inhibition and antiproliferative activity on cancer cell lines. Among fourteen tested compounds, one (compound 3b) was determined to have good activity against LAP and significant antiproliferative activity against HL-60 human promyelocytic leukemia, Burkitt's lymphoma Raji, camptothecin resistant CEM/C2 leukemia cells with mutated catalytic site of topoisomerase I, its parental cell line CCRF/CEM and LoVo colon cancer. Its influence on the cell cycle was also observed. Moreover, we have confirmed that antiproliferative activity towards cancer cells is due to LAP inhibition. Docking simulation based on positioning compound 3b into the LAP active site was performed to explore the possible binding mode. The compound was able to form hydrogen bonds with Gly362 and coordinate zinc ions, which was previously suggested to be essential for inhibitory activity. Compound 3b was also characterized with a good selectivity index for cancer versus normal mammalian cells. Toxicological studies involving examination of skin sensitization, acute skin irritation/corrosion, acute dermal toxicity, acute oral toxicity and acute eye irritation/corrosion established that compound 3b is safe for use.

1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis.

BACKGROUND: 1-methylnicotinamide (1-MNA), an endogenous metabolite of nicotinamide, has recently gained interest due to its anti-inflammatory and anti-thrombotic activities linked to the COX-2/PGI2 pathway. Given the previously reported anti-metastatic activity of prostacyclin (PGI2), we aimed to assess the effects of 1-MNA and its structurally related analog, 1,4-dimethylpyridine (1,4-DMP), in the prevention of cancer metastasis. METHODS: All the studies on the anti-tumor and anti-metastatic activity of 1-MNA and 1,4-DMP were conducted using the model of murine mammary gland cancer (4T1) transplanted either orthotopically or intravenously into female BALB/c mouse. Additionally, the effect of the investigated molecules on cancer cell-induced angiogenesis was estimated using the matrigel plug assay utilizing 4T1 cells as a source of pro-angiogenic factors. RESULTS: Neither 1-MNA nor 1,4-DMP, when given in a monotherapy of metastatic cancer, influenced the growth of 4T1 primary tumors transplanted orthotopically; however, both compounds tended to inhibit 4T1 metastases formation in lungs of mice that were orthotopically or intravenously inoculated with 4T1 or 4T1-luc2-tdTomato cells, respectively. Additionally, while 1-MNA enhanced tumor vasculature formation and markedly increased PGI2 generation, 1,4-DMP did not have such an effect. The anti-metastatic activity of 1-MNA and 1,4-DMP was further confirmed when both agents were applied with a cytostatic drug in a combined treatment of 4T1 murine mammary gland cancer what resulted in up to 80 % diminution of lung metastases formation. CONCLUSIONS: The results of the studies presented below indicate that 1-MNA and its structural analog 1,4-DMP prevent metastasis and might be beneficially implemented into the treatment of metastatic breast cancer to ensure a comprehensive strategy of metastasis control.

Bacteriophages displaying anticancer peptides in combined antibacterial and anticancer treatment.

AIMS: Novel anticancer strategies have employed bacteriophages as drug carriers and display platforms for anticancer agents; however, bacteriophage-based platforms maintain their natural antibacterial activity. This study provides the assessment of combined anticancer (engineered) and antibacterial (natural) phage activity in therapies. MATERIALS & METHODS: An in vivo BALB/c mouse model of 4T1 tumor growth accompanied by surgical wound infection was applied. The wounds were located in the areas of tumors. Bacteriophages (T4) were modified with anticancer Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides by phage display and injected intraperitoneally. RESULTS & CONCLUSION: Tumor growth was decreased in mice treated with YIGSR-displaying phages. The acuteness of wounds, bacterial load and inflammatory markers in phages-treated mice were markedly decreased. Thus, engineered bacteriophages combine antibacterial and anticancer activity.

Synthesis and antiproliferative activity of aromatic and aliphatic bis[aminomethylidene(bisphosphonic)] acids.

A series of aromatic and aliphatic bis[aminomethylidene(bisphosphonic)] acids was synthesized in the reaction of triethylphosphite with isonitriles followed by hydrolysis or dealkylation. The in vitro anti-proliferative effect of all synthesized tetraphosphonic acids against MCF-7 breast cancer cells, J774E macrophages and HL-60 promyelocytic leukemia cells was determined. Three aromatic derivatives (5a, 5f and 5j) showed a similar or higher anti-proliferative activity than zoledronic acid.

siRNA-mediated silencing of integrin ß3 expression inhibits the metastatic potential of B16 melanoma cells.

Integrins comprise a large family of αß heterodimeric cell-surface receptors that mediate diverse processes involved in cell-cell and cell-matrix interactions such as cellular adhesion and migration, cell survival and differentiation. It is now well documented that integrins play a crucial role in cancer metastasis and angiogenesis. The ß3 integrins appear to have an important stimulatory role in tumour progression and metastasis and, thus, have been often proposed as potential targets for cancer diagnosis and therapy. In this study, we evaluated the in vitro and in vivo properties of B16 mouse melanoma cells with low expression of integrin ß3. Proliferation rate, adhesive properties and the ability to migrate and metastasize were studied. Over 90% inhibition of integrin ß3 expression was achieved as a result of the transfection with siRNA. No changes in the proliferation rate were observed in siRNA-transfected B16 cells; however, they showed impaired ability to bind to fibronectin. Moreover, inhibition of integrin ß3 expression caused almost complete impairment of the ability of B16 cells to migrate through matrigel and metastasize. The mean number of lung metastatic colonies in mice inoculated intravenously with B16 expressing low levels of integrin ß3 was decreased to 14 colonies compared to 101 in the control group. These results provide evidence for a direct role of integrin ß3 in the adhesion, migration and metastasis processes of mouse melanoma cells and point to the potential therapeutic advantages of siRNAs.

The in-vitro antiproliferative effect of PRI-2191 and imatinib applied in combined treatment with cisplatin, idarubicin, or docetaxel on human leukemia cells.

Imatinib mesylate (Gleevec, STI571) is a specific inhibitor of the Bcr/Abl fusion tyrosine kinase that exhibits potent antileukemic effects in chronic myelogenous leukemia. Bcr/Abl-positive K562 and Bcr/Abl-negative HL-60 human leukemia cells were used to investigate the effect of PRI-2191, a calcitriol analog, on the biological effects of imatinib combined with other anticancer drugs. The results show that PRI-2191 enhances the antiproliferative effect of imatinib on HL-60 cells. When these two agents together are applied with either docetaxel or cisplatin, but not with idarubicin, the antiproliferative effect could still be enhanced. Moreover, when the interaction between the chemotherapy agents was antagonistic or additive, PRI-2191 could even shift it to synergism. This effect correlated with an accumulation of HL-60 cells in the G0/G1 phase of the cell cycle and a decrease in the percentage of cells in the G2/M and S stage in the ternary combinations used. PRI-2191 did not influence apoptosis induced by imatinib alone or in ternary combinations with all the chemotherapy agents used. These results may suggest that the stronger antiproliferative effect of the combined treatment with PRI-2191 on HL-60 cells is related to cell cycle arrest rather than to the induction of apoptosis.

Insights into the mechanisms involved in magnesium-dependent inhibition of primary tumor growth.

We have previously shown that a low Magnesium (Mg)-containing diet reversibly inhibits the growth of primary tumors that develop after the injection of Lewis lung carcinoma (LLC) cells in mice. Here we investigate some of the mechanisms responsible for the Mg-dependent regulation of tumor development by studying cell cycle regulation, tumor angiogenesis, and gene expression under Mg deficiency. The inhibition of LLC tumor growth in Mg-deficient mice is due to a direct effect of low Mg on LLC cell proliferation and to an impairment of the angiogenic switch. We also observed an increase of nitric oxide synthesis and oxidative DNA damage. Complementary DNA arrays reveal that Mg deficiency modulates tumor expression of genes involved in the control of cell cycle, stress response, proteolysis, and adhesion. Our results suggest that Mg has multiple and complex roles in tumor development.