ABSTRACT
Our research found that vitamin D3 (VD3) treatment increased lung metastasis in mice with 4T1 murine breast cancer (BC). This study aims to investigate the impact of VD3 on the activation of tumor-associated macrophages (TAMs) in BC. Mice bearing 4T1, E0771, 67NR BC cells, and healthy mice, were fed diets with varying VD3 contents (100-deficient, 1000-normal, and 5000 IU/kg-elevated). Some mice in the 1000 and 100 IU/kg groups received calcitriol. We studied bone metastasis and characterized TAMs and bone marrow-derived macrophages (BMDMs). 4T1 cells had higher bone metastasis potential in the 5000 IU/kg and calcitriol groups. In the same mice, an elevated tumor osteopontin level and M2 polarization of TAMs (MHCIIlow CD44high phenotype) were observed. Gene expression analysis confirmed M2 polarization of 4T1 (but not 67NR) TAMs and BMDMs, particularly in the 100 IU + cal group (increased Mrc1, Il23, and Il6). This polarization was likely due to COX-2/PGE2 induction in 4T1 calcitriol-treated cells, leading to increased proinflammatory cytokines like IL-6 and IL-23. Future studies will explore COX-2/PGE2 as a primary mediator of calcitriol-stimulated inflammation in the BC microenvironment, especially relevant for BC patients with VD3 deficiency and supplementation.
Subject(s)
Breast Neoplasms , Mammary Glands, Human , Humans , Animals , Mice , Female , Cytokines/metabolism , Calcitriol/pharmacology , Tumor-Associated Macrophages/metabolism , Cyclooxygenase 2/genetics , Mammary Glands, Human/metabolism , Cell Line, Tumor , Breast Neoplasms/pathology , Tumor MicroenvironmentABSTRACT
BACKGROUND: Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Despite the well-known in vitro antitumoral effect of vitamin D3 (VD3), its impact on breast CAFs is almost unknown. In this study, we analyzed the ex vivo effects of calcitriol on CAFs isolated from breast cancer tissues. METHODS: CAFs were cultured with 1 and 10 nM calcitriol and their phenotype; gene expression, protein expression, and secretion were assessed. Calcitriol-treated CAFs-conditioned media (CM) were used to analyze the effect of CAFs on the migration and protein expression of MCF-7 and MDA-MB-231 cells. RESULTS: Tumor tissues from VD3-deficient patients exhibited lower levels of ß-catenin and TGFß1, along with higher levels of CYP24A1 compared to VD3-normal patients. In VD3-deficient patients, CAF infiltration was inversely associated with CYP24A1 levels and positively correlated with OPN levels. Calcitriol diminished CAFs' viability, but this effect was weaker in premenopausal and VD3-normal patients. Calcitriol reduced mRNA expression of CCL2, MMP9, TNC, and increased PDPN, SPP1, and TIMP1. It also decreased the secretion of CCL2, TNC, and the activity of MMP-2, while increasing cellular levels of TIMP1 in CAFs from all patient groups. In nonmetastatic and postmenopausal patients, PDPN surface expression increased, and CAFs CM from these groups decreased MCF-7 cell migration after ex vivo calcitriol treatment. In premenopausal and VD3-deficient patients, calcitriol reduced IDO1 expression in CAFs. Calcitriol-treated CAFs CM from these patients decreased OPN expression in MCF-7 and/or MDA-MB-231 cells. However, in premenopausal patients, calcitriol-treated CAFs CM also decreased E-cadherin expression in both cell lines. CONCLUSION: The effects of calcitriol on breast CAFs, both at the gene and protein levels, are complex, reflecting the immunosuppressive or procancer properties of CAFs. The anticancer polarization of CAFs following ex vivo calcitriol treatment may result from decreased CCL2, TNC (gene and protein), MMP9, and MMP-2, while the opposite effect may result from increased PDPN, TIMP1 (gene and protein), and SPP1. Despite these multifaceted effects of calcitriol on molecule expression, CAFs' CMs from nonmetastatic and postmenopausal patients treated ex vivo with calcitriol decreased the migration of MCF-7 cells.
Subject(s)
Breast Neoplasms , Cancer-Associated Fibroblasts , Humans , Female , Cancer-Associated Fibroblasts/metabolism , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase 2/metabolism , Vitamin D3 24-Hydroxylase/genetics , Vitamin D3 24-Hydroxylase/metabolism , Cholecalciferol , Calcitriol/pharmacology , Fibroblasts/metabolism , Cell Movement/genetics , Cell Line, Tumor , Tumor Microenvironment/geneticsABSTRACT
Background: MALAT1 is one of the most abundant nuclear long non-coding RNAs, which has been found to be elevated in various types of cancers. However, conflicting reports on MALAT1 in breast cancer cell lines challenge understanding of MALAT1's involvement in breast cancer progression. Aim: Measurement of normalized relative quantity (NRQ) of MALAT1 transcripts in cell lines representing triple-negative breast cancer (TNBC) and luminal breast cancer. Materials and methods: The studies were performed using cell lines representing luminal breast cancer (T47D, MCF-7), TNBC (MDA-MB-468, CAL-51, MDA-MB-231), and MCF-10A cell line of normal breast epithelial cells. Total RNA was isolated from six independent cell cultures of each line, treated with DNase I, and used to synthesize complementary DNA, which was used in quantitative real-time PCR (qPCR) assays. Four MALAT1 fragments and reference genes CCSER2, ANKRD17, PUM1, GAPDH were amplified. Results: Geometric means of the NRQ of MALAT1 in breast cancer cell lines had the shortest 95% confidence intervals when CCSER2 was used for normalization. MALAT1 major transcript levels thus estimated in TNBC cell lines were found to be statistically significantly reduced compared to levels in both MCF-10A cells and luminal breast cancer cell lines, while MALAT1 minority splice variants were found to be increased in almost all breast cancer cell lines. Conclusion: CCSER2-normalized qPCR results indicate MALAT1 downregulation in cell lines representing the more aggressive breast cancer subtype compared to both the normal breast epithelial cell line and the estrogen receptor-positive breast cancer cell lines.
ABSTRACT
Non-coding micro-RNA (miRNAs) regulate the protein expression responsible for cell growth and proliferation. miRNAs also play a role in a cancer cells' response to drug treatment. Knowing that leukemia and lymphoma cells show different responses to active forms of vitamin D3, we decided to investigate the role of selected miRNA molecules and regulated proteins, analyzing if there is a correlation between the selected miRNAs and regulated proteins in response to two active forms of vitamin D3, calcitriol and tacalcitol. A total of nine human cell lines were analyzed: five leukemias: MV-4-1, Thp-1, HL-60, K562, and KG-1; and four lymphomas: Raji, Daudi, Jurkat, and U2932. We selected five miRNA molecules-miR-27b, miR-32, miR-125b, miR-181a, and miR-181b-and the proteins regulated by these molecules, namely, CYP24A1, Bak1, Bim, p21, p27, p53, and NF-kB. The results showed that the level of selected miRNAs correlates with the level of proteins, especially p27, Bak1, NFκB, and CYP24A1, and miR-27b and miR-125b could be responsible for the anticancer activity of active forms of vitamin D3 in human leukemia and lymphoma.
Subject(s)
Cholecalciferol , Leukemia , Lymphoma , MicroRNAs , Cell Line/drug effects , Cell Line/metabolism , Cell Proliferation , Cholecalciferol/pharmacology , Humans , Leukemia/genetics , Leukemia/metabolism , Lymphoma/genetics , Lymphoma/metabolism , MicroRNAs/drug effects , MicroRNAs/genetics , MicroRNAs/metabolism , Vitamin D3 24-HydroxylaseABSTRACT
The active forms of vitamin D3 (calcitriol and tacalcitol) coupled to the vitamin D receptor (VDR) are known to exhibit anti-cancer properties. However, not all cancer cells are sensitive to the active forms of vitamin D3 and its analogs. The study aimed to determine whether polymorphism of VDR is responsible for the sensitivity of human leukemia and lymphoma cells to calcitriol and tacalcitol. The impact of calcitriol and tacalcitol on the proliferation and morphology of nine different leukemia and lymphoma cell lines was determined. Only MV-4-11, Thp-1, and HL-60 cell lines sensitive to proliferation inhibition by calcitriol and tacalcitol showed morphology changes. Subsequently, the levels of the VDR and 1,25D3-MARRS proteins of calcitriol and tacalcitol binding receptors and the VDR receptor polymorphism in human leukemia and lymphoma cells were ascertained. Contrary to the current understanding, higher levels of VDR are not responsible for the greater sensitivity of cells to calcitriol and tacalcitol. Importantly, we first showed that sensitivity to calcitriol and tacalcitol in leukemias and lymphomas could be determined by the VDR polymorphism. The FokI polymorphism and the presence of the "bat" haplotype were observed only in the sensitive cells.
ABSTRACT
A library of 21 novel chiral 1,2,3-triazole-based 2-azabicycloalkane conjugates was designed and synthesized using the copper(I)-catalyzed click reaction. The obtained hybrids were assessed for their antiproliferative potency against three selected human cancer cell lines: Hs294T (melanoma), MIA PaCa-2 (pancreas tumor) and NCI-H1581 (lung tumor). The majority of the synthesized compounds demonstrated moderate to potent activity, and some of them appeared more selective than cisplatin, with selectivity index exceeding 9.
ABSTRACT
To analyze if the prometastatic activity of calcitriol (active vitamin D3 metabolite), which was previously observed in a 4T1 breast cancer model, is also found in other breast cancers, and to assess the impact of various schemes of vitamin D supply, we used 4T1 and E0771 mouse metastatic and 67NR nonmetastatic cells in this study. BALB/c and C57BL/6 healthy and tumor-bearing mice were exposed to a control (1000 IU), low- (100 IU), and high- (5000 IU) vitamin D3 diets. Additionally, from day 7 of tumor transplantation, the 1000 and 100 IU groups were gavaged with calcitriol (+cal). After 8 weeks of feeding, plasma levels of 25(OH)D3, 24,25(OH)2D3, and 3-epi-25(OH)D3 were significantly lower in calcitriol-treated and vitamin D-deficient groups than in the control, whereas the levels of all metabolites were increased in the 5000 IU group. The ratio of 25(OH)D3:24,25(OH)2D3 was increased in both calcitriol-treated groups, whereas the ratio of 25(OH)D3:3-epi-25(OH)D3 was increased only in the 100 IU group but decreased in the 5000 IU group. In contrast to E0771, 4T1 lung metastasis was accelerated in all vitamin D-supplemented mice, as well as in the deficient group with an increased inflammatory response. 67NR tumor growth was transiently inhibited in the 1000 IU+cal group, but single metastases were observed in the 5000 and 100 IU groups. Based on the results, we conclude that various schemes of vitamin D supply and vitamin D deficiency led to similar metabolite profiles irrespective of the mice strain and tumor burden. However, depending on the type of breast cancer, different effects on tumor growth and metastasis were noticed.
Subject(s)
Calcitriol/therapeutic use , Cholecalciferol/therapeutic use , Dietary Supplements , Mammary Neoplasms, Animal/drug therapy , Metabolome , Vitamin D/metabolism , Animals , Body Weight/drug effects , Calcitriol/pharmacology , Cholecalciferol/pharmacology , Female , Kidney/metabolism , Kinetics , Liver/metabolism , Lung Neoplasms/secondary , Mammary Neoplasms, Animal/blood , Mammary Neoplasms, Animal/pathology , Mice, Inbred BALB C , Mice, Inbred C57BL , Models, Biological , Neoplasm Proteins/metabolism , Vitamin D/bloodABSTRACT
Consuming food that is rich in antioxidants reduces the risk of developing chronic diseases and oxidative stress. Fruits and vegetables are an excellent source of substances with antioxidant and pro-health properties. Such raw materials, characterized by a high content of polyphenolic compounds and antioxidant capacity, include pear fruits. In this study, the concentrations of bioactive compounds, as well as the antioxidant, anti-inflammatory, and antiproliferative activity in fruits of five selected pear cultivars were determined and compared. LC-MS and UPLC-PDA methods were used to determine the polyphenolic, carotenoid, chlorophyll, and triterpenoid profiles and content, and the antioxidant activity was analyzed using DPPH and ferric-reducing ability of plasma (FRAP) tests. The anti-inflammatory activity was determined against COX-1 and COX-2 enzymes. The cytotoxic activity of the test compounds was assessed against six tumor cell lines. The results showed that the major group of phenolic compounds in all cultivars was phenolic acids. In the group of chromoplastic pigments, chlorophyllide a and 9-cis-ß-carotene were the major compounds, while in the triterpene group, ursolic acid was dominant. The antioxidant potential correlated with the content of polyphenols and carotenoids, and was the strongest for the 'Radana' cultivar. The highest antiproliferative activity in all varieties was established for bladder cancer.
Subject(s)
Antineoplastic Agents, Phytogenic , Antioxidants , Cytotoxins , Fruit/chemistry , Neoplasms/drug therapy , Polyphenols , Pyrus/classification , A549 Cells , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Antioxidants/chemistry , Antioxidants/pharmacology , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Cytotoxins/chemistry , Cytotoxins/pharmacology , HT29 Cells , Humans , MCF-7 Cells , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Polyphenols/chemistry , Polyphenols/pharmacologyABSTRACT
Vitamin D and its analogs are known for their role in the development of breast cancer and in immunomodulation. Our previous studies have shown the pro-metastatic effect of calcitriol and tacalcitol (PRI-2191) in young mice bearing 4T1 breast cancer and the anti-metastatic effect in aged ovariectomized (OVX) mice. Therefore, the aim of our work was to characterize Th17 cell population in young and aged OVX mice bearing 4T1 tumors treated with calcitriol and PRI-2191. The expression of genes typical for Th17 cells was examined in splenocytes, as well as splenocytes differentiated with IL-6 and TGF-ß to Th17 cells (iTh17). Expression of genes encoding vitamin D receptor (Vdr) and osteopontin (Spp1) as well as the secretion of IL-17A were evaluated in iTh17 cells. PRI-2191 treatment increased the expression of Rora and Rorc transcription factors, Il17a, Il17re and Il21 in iTh17 cells from young mice. In aged OVX mice this effect was not observed. Increased expression was observed in the case of Vdr and Spp1 genes in iTh17 cells from young mice treated with PRI-2191. What is more, in young mice treated with PRI-2191 the secretion of IL-17A to the culture media by iTh17 cells was increased, whereas in aged OVX mice a significant decrease was noted. Increased expression of Spp1 in young mice treated with PRI-2191 may enhance the differentiation of Th17 cells.
ABSTRACT
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.
ABSTRACT
The process of apoptosis begins when the balance between proapoptotic and antiapoptotic stimuli is disturbed, leading to oligomerization of apoptosis effectors and disruption of the outer mitochondrial membrane. BCL-2 family proteins are the major regulators of mitochondrial pathway of apoptosis. In turn, microRNA-125b (miR-125b) is a member of microRNAs, which are short single-stranded noncoding RNAs that negatively regulate gene expression at the posttranscriptional level. miR-125b targets messenger RNAs encoding proapoptotic (BAK1, PUMA, BMF) and antiapoptotic (MCL1) BCL-2 family proteins. This mini-review briefly describes the involvement of BCL-2 family proteins in triggering apoptosis. Then, attention is paid to the differences in the activation of apoptosis with doxorubicin and paclitaxel, and finally the effect of miR-125b on paclitaxel- and doxorubicin-induced apoptosis in breast cancer cells is considered. It appears that miR-125b downregulates proteins that are significantly involved in the activation of apoptosis after paclitaxel-induced prolonged mitotic arrest or subsequent DNA damage if mitotic slippage occurs. It seems that high levels of miR-125b may not favor paclitaxel therapy, but reduction of miR-125b levels may increase paclitaxel-induced apoptosis, possibly also genotoxic-induced apoptosis, although adverse effects may also occur including decrease in doxorubicin-induced apoptosis.
Subject(s)
Apoptosis/genetics , Breast Neoplasms/genetics , MicroRNAs/genetics , Mitosis/genetics , Animals , Female , Humans , Mitochondria/genetics , Proto-Oncogene Proteins c-bcl-2/geneticsABSTRACT
BACKGROUND/AIM: The study aimed to test the potential for increasing the antiproliferative activity of 5-fluorouracil against breast cancer cells of various molecular subtypes by vitamin D receptor (VDR) agonists, calcitriol and tacalcitol, used at a low concentration of 10 nM. MATERIALS AND METHODS: Calcitriol and tacalcitol were used to increase the antiproliferative effect of 5-fluorouracil against the following human breast cancer cell lines: MCF-7, T47D, BT-474 (luminal); JIMT-1, SKBR-3 (HER2-enriched); MDA-MB-231 (triple-negative/basal-B), and non-malignant MCF-10A breast epithelial cells. RESULTS: Both calcitriol and tacalcitol significantly increased the sensitivity of MCF-7 and BT-474 cells to the antiproliferative effect of 5-fluorouracil, while no increase in the sensitivity of MDA-MB-231 cells to 5-fluorouracil treatment was observed. CONCLUSION: The VDR agonist used at the relatively low concentration of 10 nM may increase the sensitivity of breast cancer cells, at least of the luminal subtype, to the antiproliferative effect of 5-fluorouracil.
Subject(s)
Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Fluorouracil/therapeutic use , Receptors, Calcitriol/agonists , Cell Line, Tumor , Fluorouracil/pharmacology , Humans , MCF-7 Cells/metabolismABSTRACT
5-Fluorouracil (5-FU) is an anticancer drug that is most frequently used to treat colorectal cancer (CRC) patients, but unfortunately it shows limited efficacy. We recently demonstrated that vitamin D analogs (VDAs), particularly tacalcitol (coded as PRI-2191), potentiate its anticancer activity in an in vivo mouse and human CRC model. The purpose of this study was to explain the mechanism underlying the enhancement of 5-FU efficacy by PRI-2191 towards human HT-29 CRC cells. We showed that PRI-2191 induces the CDKN1A (gene encoding p21Waf1/Cip1) expression directly through vitamin D receptor (VDR) in a p53-independent manner and thus decreases the thymidylate synthase expression both at the mRNA and protein level. It is the main mechanism by which PRI-2191 improves the anticancer efficacy of 5-FU towards HT-29 cells. Additionally, we indicated that the VDR also participates in 5-FU mechanism of action. 5-FU significantly increased TYMS (gene encoding thymidylate synthase (TS)) and BIRC5 (gene encoding survivin) level in HT-29 cells with silenced VDR. Furthermore, PRI-2191 induced E-cadherin and ZO-1 expression and thus reduced the level of BIRC5 in HT-29 cells. The induction of E-cadherin expression may also contribute to the reduction of c-Myc level and consequently the downregulation of TS. Our results also indicate that calcium-sensing receptor (CaSR) plays a role in the activity of PRI-2191 but has no influence on the 5-FU mechanism of action. In conclusion, we suggest that both VDR and CaSR might be useful as molecular markers for predicting treatment outcomes and identifying the CRC patient subgroups who might benefit from 5-FU-based chemotherapy combined with vitamin D analog.
Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Colorectal Neoplasms/drug therapy , Dihydroxycholecalciferols/pharmacology , Fluorouracil/pharmacology , Thymidylate Synthase/genetics , Colorectal Neoplasms/genetics , Down-Regulation/drug effects , Drug Resistance, Neoplasm/drug effects , Drug Synergism , HT29 Cells , HumansABSTRACT
MiR-125b belongs to the class of microRNAs, which are short endogenous non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. Recently, it was reported that miR-125b was found to promote migration and invasion of MCF-7 cells and was involved in chemotherapeutic resistance. Decreasing miR-125b expression would have potential therapeutic significance in preventing dissemination of breast cancer cells. The objective of this study was to evaluate miR-125b expression levels in MCF-7 cells following treatment with 1,25-dihydroxyvitamin D3 (calcitriol) and 1,24-dihydroxyvitamin D3 (tacalcitol), active metabolite and synthetic analog of vitamin D3, respectively. We found that treatment with both calcitriol and tacalcitol caused a decrease in miR-125b expression. In addition, treatment with calcitriol and tacalcitol resulted in an increase in the level of pro-apoptotic BAK1 protein encoded by the target gene of miR-125b. We are discussing the putative mechanism of inhibition of the miR-125b expression by vitamin D receptor (VDR) agonists and we suggest that calcitriol and tacalcitol may be used as a miR-125b inhibitor in breast cancer cells expressing VDR.
Subject(s)
Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Calcitriol/pharmacology , Dihydroxycholecalciferols/pharmacology , MicroRNAs/genetics , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , MCF-7 Cells , MicroRNAs/biosynthesis , Molecular Conformation , Reverse Transcriptase Polymerase Chain Reaction , Structure-Activity RelationshipABSTRACT
Low vitamin D status is considered as a risk factor for breast cancer and has prognostic significance. Furthermore, vitamin D deficiency increases after adjuvant cancer therapy, which alters bone metabolism increasing the risk of osteoporosis. It is now postulated that vitamin D supplementation in breast cancer treatment delays the recurrence of cancer thereby extending survival. We evaluated the impact of calcitriol and its low-calcemic analogs, PRI2191 and PRI2205, on the tumor growth, angiogenesis, and metastasis of 4T1 mouse mammary gland cancer. Gene expression analysis related to cancer invasion/metastasis, realtime PCR, ELISA, western blotting, and histochemical studies were performed. In vitro studies were conducted to compare the effects of calcitriol and its analogs on 4T1 and 67NR cell proliferation and expression of selected proteins. Calcitriol and its analogs increased lung metastasis without influencing the growth of primary tumor. The levels of plasma 17ß-estradiol and transforming growth factor ß (TGFß) were found to be elevated after treatment. Moreover, the results showed that tumor blood perfusion improved and osteopontin (OPN) levels increased, whereas vascular endothelial growth factor (VEGF) and TGFß levels decreased in tumors from treated mice. All the studied treatments resulted in increased collagen content in the tumor tissue in the early step of tumor progression, and calcitriol caused an increase in collagen content in lung tissue. In addition, in vitro proliferation of 4T1 tumor cells was not found to be affected by calcitriol or its analogs in contrast to non-metastatic 67NR cells. Calcitriol and its analogs enhanced the metastatic potential of 4T1 mouse mammary gland cancer by inducing the secretion of OPN probably via host cells. In addition, OPN tumor overexpression prevailed over the decreasing tumor TGFß level and blood vessel normalization via tumor VEGF deprivation induced by calcitriol and its analogs. Moreover, the increased plasma TGFß and 17ß-estradiol levels contributed to the facilitation of metastatic process.
Subject(s)
Calcitriol/analogs & derivatives , Calcitriol/pharmacology , Dihydroxycholecalciferols/pharmacology , Mammary Neoplasms, Experimental/blood supply , Mammary Neoplasms, Experimental/pathology , Animals , Cell Growth Processes/drug effects , Cell Line, Tumor , Female , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/genetics , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Real-Time Polymerase Chain Reaction , Tumor Microenvironment/drug effectsABSTRACT
The paper shows that new N'-substituted 2,4-dihydroxybenzocarbothiohydrazides are able to inhibit the in vitro proliferation of human tumor cell lines. The compounds were prepared by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione] (STB) or its analogs with the hydrazines. The panel of N'-substitution included aryl, pyridinyl and pyrimidinyl rings. The highest antiproliferative activity for N'-(4-(4-chlorophenyl)-6-(trifluoromethyl)pyrimidin-2-yl)-5-ethyl-2,4-dihydroxybenzothiohydrazide (5b) was found. The antiproliferative potency of some compounds was similar to that of cisplatin. Analogs with the Et substituent on benzenediol moiety displayed higher potency than with the unsubstituted one. The influence of N'-substitution on antiproliferative activity of compounds was discussed.
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Hydrazines/chemical synthesis , Hydrazines/pharmacology , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Hydrazines/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
The rat PC12 cell line has become a widely used research tool for many aspects of neurobiology. Nerve growth factor (NGF)-responsive PC12 cells were engineered to drive expression of doxycycline (Dox)-induced gene of interest in the Tet-On expression system that resulted in obtaining PC12-Tet-On cells. TrkA and TrkC are neurotrophin receptors derived from the tropomyosin-related kinase (Trk) family of receptor tyrosine kinases. TrkA receptor binds and is activated mainly by NGF, while TrkC receptor binds and is activated by neurotrophin 3 (NT3). The purpose of this research was to design and describe PC12-based neuronal cell model to study TrkC-triggered versus TrkA-triggered neurite outgrowth. The second-generation tetracycline-responsive promoter (P tight) was used in order to provide low basal expression in the absence of Dox and high-level Dox-induced expression of TrkC. The main advantage of presented model system is dependence of TrkC level on Dox concentration. It also allows to compare activation of intracellular signaling proteins and neurite outgrowth following activation of TrkA and TrkC receptors by NGF and NT3, respectively, in the context of the same quality and quantity of intracellular adaptor proteins, Ras proteins, protein kinases and phosphatases, and phospholipase Cγ1, as a difference in the activation of intracellular signaling network by these two distinct although related receptor tyrosine kinases is expected. The results of our studies suggest that despite slightly weaker activation of ERK1/2 mitogen-activated protein kinases, NT3-triggered TrkC seems to provide apparently stronger than NGF-triggered TrkA signal for neurite elongation in differentiating PC12 cells.
Subject(s)
Models, Biological , Neurites/metabolism , Neurogenesis , Receptor, trkC/metabolism , Animals , Doxycycline/pharmacology , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Neurites/drug effects , Neurogenesis/drug effects , Neurotrophin 3/pharmacology , PC12 Cells , Rats , TransfectionABSTRACT
Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.
Subject(s)
Adenosine/pharmacology , Adrenal Gland Neoplasms/enzymology , Adrenal Gland Neoplasms/genetics , Cyclic Nucleotide Phosphodiesterases, Type 2/metabolism , Pheochromocytoma/genetics , Transcription, Genetic/drug effects , Tyrosine 3-Monooxygenase/genetics , Animals , Atrial Natriuretic Factor/pharmacology , Cyclic AMP/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclic GMP/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Models, Biological , PC12 Cells , Pheochromocytoma/enzymology , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Oxazolinodoxorubicin, a doxorubicin analog with a modified daunosamine moiety was synthesized. The properties of this compound and the parent doxorubicin were compared. The cytotoxicity in vitro studies against several human tumor cell lines (PC-3, MCF-7, SW707, HL-60, RPMI 8226, ACHN) showed higher antiproliferative potency for this new compound. Moreover, its ability to completely overcome the drug resistance of cancer cells in vitro was revealed (LoVo, LoVo/DX, MES-SA, MES-SA/DX5, HL-60, HL-60/Vinc, HL-60/MX2 cell lines). Cellular uptake analyzed on HL-60 and HL-60/MX2 cells, demonstrated higher penetration levels of oxazolinodoxorubicin compared to that of doxorubicin. In animal experiments, general toxicity of oxazolinodoxorubicin was lower than that observed for doxorubicin. Furthermore, similar antitumor effects was observed in NOD/SCID mice bearing resistant HL-60/Vinc leukemia tumor and in mice treated with the new or parent compounds. The presented results suggest that oxazolinodoxorubicin is a new anthracycline with an advantageous biological activity profile.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Daunorubicin/analogs & derivatives , Doxorubicin/analogs & derivatives , Animals , Antibiotics, Antineoplastic/chemical synthesis , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacokinetics , Body Weight/drug effects , Cell Line, Tumor , Daunorubicin/chemical synthesis , Daunorubicin/chemistry , Daunorubicin/pharmacokinetics , Daunorubicin/pharmacology , Doxorubicin/pharmacology , Female , HL-60 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Mice, Inbred NOD , Mice, SCID , Xenograft Model Antitumor AssaysABSTRACT
A one-pot synthesis of new 4-(1,3-thiazolo[5,4-b]pyridin-2-yl)benzene-1,3-diols has been described. The compounds were prepared by the reaction of sulfinylbis[(2,4-dihydroxyphenyl)methanethione] derivatives, with various substituents in the aryl rings, with 2-chloropyridin-3-amines. Their structures were deduced from IR and, (1) H- and (13) C-NMR spectroscopic, mass spectrometric, and elemental analyses. The antiproliferative properties of some of the products against human cancer cell lines were comparable to those of cisplatin. Structure-activity analysis showed that the presence of hydrophobic substituents in both heterocyclic fused and phenyl rings of the compounds improves their biological effects. Further, an additional OH group in the resorcinol moiety reduced the antiproliferative activity.