Celecoxib alleviates tamoxifen-instigated angiogenic effects by ROS-dependent VEGF/VEGFR2 autocrine signaling

Background: Tamoxifen (TAM) is widely used in the chemotherapy of breast cancer and as a preventive agent against recurrence after surgery. However, extended TAM administration for breast cancer induces increased VEGF levels in patients, promoting new blood vessel formation and thereby limiting its efficacy. Celecoxib (CXB), a selective COX-2 inhibitor, suppresses VEGF gene expression by targeting the VEGF promoter responsible for its inhibitory effect. For this study, we had selected CXB as non-steroidal anti-inflammatory drug in combination with TAM for suppressing VEGF expression and simultaneously reducing doses of both the drugs. Methods: The effects of CXB combined with TAM were examined in two human breast cancer cell lines in culture, MCF7 and MDA-MB-231. Assays of proliferation, apoptosis, angiogenesis, metastasis, cell cycle distribution and receptor signaling were performed. Results: Here, we elucidated how the combination of TAM and CXB at nontoxic doses exerts anti-angiogenic effects by specifically targeting VEGF/VEGFR2 autocrine signaling through ROS generation. At the molecular level, TAM-CXB suppresses VHL-mediated HIF-1α activation, responsible for expression of COX-2, MMP-2 and VEGF. Besides low VEGF levels, TAM-CXB also suppresses VEGFR2 expression, confirmed through quantifying secreted VEGF levels, luciferase and RT-PCR studies. Interestingly, we observed that TAM-CXB was effective in blocking VEGFR2 promoter induced expression and further 2 fold decrease in VEGF levels was observed in combination than TAM alone in both cell lines. Secondly, TAM-CXB regulated VEGFR2 inhibits Src expression, responsible for tumor progression and metastasis. FACS and in vivo enzymatic studies showed significant increase in the reactive oxygen species upon TAM-CXB treatment. Conclusions: Taken together, our experimental results indicate that this additive combination shows promising outcome in anti-metastatic and apoptotic studies. In a line, our preclinical studies evidenced that this additive combination of TAM and CXB is a potential drug candidate for treatment of breast tumors expressing high levels of VEGF and VEGFR2. This ingenious combination might be a better tailored clinical regimen than TAM alone for breast cancer treatment

Novel ZnO hollow-nanocarriers containing paclitaxel targeting folate-receptors in a malignant pH-microenvironment for effective monitoring and promoting breast tumor regression

Low pH in the tumor micromilieu is a recognized pathological feature of cancer. This attribute of cancerous cells has been targeted herein for the controlled release of chemotherapeutics at the tumour site, while sparing healthy tissues. To this end, pH-sensitive, hollow ZnO-nanocarriers loaded with paclitaxel were synthesized and their efficacy studied in breast cancer in vitro and in vivo. The nanocarriers were surface functionalized with folate using click-chemistry to improve targeted uptake by the malignant cells that over-express folate-receptors. The nanocarriers released ~75% of the paclitaxel payload within six hours in acidic pH, which was accompanied by switching of fluorescence from blue to green and a 10-fold increase in the fluorescence intensity. The fluorescence-switching phenomenon is due to structural collapse of the nanocarriers in the endolysosome. Energy dispersion X-ray mapping and whole animal fluorescent imaging studies were carried out to show that combined pH and folate-receptor targeting reduces off-target accumulation of the nanocarriers. Further, a dual cell-specific and pH-sensitive nanocarrier greatly improved the efficacy of paclitaxel to regress subcutaneous tumors in vivo. These nanocarriers could improve chemotherapy tolerance and increase anti-tumor efficacy, while also providing a novel diagnostic read-out through fluorescent switching that is proportional to drug release in malignant tissues

Prevalence of thyroid dysfunction among women during the first trimester of pregnancy at a tertiary care hospital in Haryana

Background: Undetected and untreated thyroid disorders are associated with adverse maternal and fetal outcomes. There are limited data on the prevalence of newly diagnosed thyroid disease during pregnancy from India. Therefore, this study was designed to evaluate the prevalence of thyroid dysfunction, especially hypothyroidism during the first trimester of pregnancy. Materials and Methods: The present cross-sectional study was conducted at Department of endocrinology and antenatal clinic in the Obstetrics and Gynecology Pt. B.D. Sharma PGIMS, Rohtak over a period of 1-year. The total sample population comprised of 461 pregnant women with uncomplicated intrauterine singleton pregnancies in the first trimester of gestation without any history of thyroid disease or intake of any thyroid medication. Morning blood samples from the participants were analyzed for thyroid function tests, which included FT3, FT4, thyroid-stimulating hormone (TSH) and anti-thyroid peroxidase antibodies (TPO). Results: A total of 461 women were enrolled for this study. Mean maternal age was 23.79 ± 3.47 years. Median gestational age was 8 weeks 5 days. The median FT3, FT4 and TSH were 3.3 pg/mL, 1.25 ng/dL, and 1.40 mIU/L, respectively. Anti-TPO was elevated in 128 (27.8%) pregnant women. 99 (21.5%) women had sub-clinical hypothyroidism and 39 (39.4%) among them were positive for anti-TPO ( P ≤ 0.001). 2 (0.4%) of women had overt hyperthyroidism, whereas 15 (3.3%) of the women had sub-clinical hyperthyroidism. Conclusion: Considering the immense impact that maternal thyroid dysfunction has on maternal and fetal outcomes, prompt identification of thyroid dysfunction and its timely treatment is essential. Thus, universal screening of pregnant women for thyroid dysfunction should be considered especially in a country like India due to the high prevalence of thyroid dysfunction

Electric cell–substrate impedance sensing technique to monitor cellular behaviours of cancer cells

The present work reports the cellular electrical behaviour of the MDA-MB-231 breast cancer cell line treated with the anticancer drug ZD6474, using impedance sensing devices. Microelectrode-based devices with four different electrode geometries are fabricated by microfabrication technology. Real-time impedance monitoring data show high impedance variation during the initial 5 hours, revealing rapid spreading of cells over electrode surfaces. It is further established that impedance variation is mostly controlled by cells covering the electrode surface area, and thus, an enhanced effect is seen with electrode devices with a smaller geometry. Real-time impedimetric cytotoxicity data reveal that cell death and detachment starts at 21 h after inoculation of cells in the devices. The frequency response characteristics of drug-treated cells are studied to evaluate the cytotoxic effect of ZD6474. Compared to the control, a significant variation in the magnitude of the measured impedance data is observed for drug-treated samples above a 5 μM dose, indicating cell growth suppression and cell death. Finally, an empirical relationship between cell impedance and drug dose is established from impedance data, which shows that they are negatively correlated

Frequency dependent impedimetric cytotoxic evaluation of anticancer drug on breast cancer cell

The present work reports the impedance characteristics of MCF-7 cell lines treated with anticancer drug ZD6474 to evaluate the cytotoxic effect on cellular electrical behaviour using miniature impedance sensors. Four types of impedance sensing devices with different electrode geometries are fabricated by microfabrication technology. The frequency response characteristics of drug treated cells are studied to evaluate cytotoxic effect of ZD6474 and also to assess the frequency dependent sensitivity variation with electrode area. A significant variation in magnitude of measured impedance data is obtained for drug treated samples above 10 μM dose indicating prominent effect of ZD6474 which results in suppression of cell proliferation and induction of apoptosis process. The results obtained by impedimetric method are correlated well with conventional in vitro assays such as flow cytometry, cell viability assays and microscopic imaging. Finally an empirical relation between cell impedance, electrode area and drug dose is established from impedance data which exhibits a negative correlation between drug doses and impedance of cancer cells

BI2536 – A PLK inhibitor augments paclitaxel efficacy in suppressing tamoxifen induced senescence and resistance in breast cancer cells

Tamoxifen resistance is a multifaceted phenomenon, characterized by the constitutive activation of multiple signaling cascades that provide an additional survival advantage to cells. Ground studies related to reverse the tamoxifen resistance by employing chemotherapeutic drugs that specifically inhibit proteins, those of aberrantly expressed, are required. Seminal studies showed that p38 signaling and VEGF play crucial role in acquiring resistance to tamoxifen. In this view, we had chosen paclitaxel, a mitotic inhibitor with anti-proliferative effects against a wide array of cancers in this study. Further to mitigate the undesirable complications of Paclitaxel (PAC), we employed this drug in combination along with BI2536 (BI), a PLK inhibitor for this study to sensitize the tamoxifen resistant cells to apoptosis. MCF 7/TAM and T-47D/TAM cells were treated with PAC, BI and in combination (BI-PAC) evaluated for its anticancer activity through apoptotic and western blot analysis. Modulatory effects of BI-PAC on p38 inactivation were affirmed through immunofluorescence and drug potential studies. Results reveal that cells were subjected to apoptosis on drug(s) treatment which was confirmed through cytotoxicity, annexin studies. Further, the anti-proliferative effects of the drug(s) were affirmed through nuclear morphological and TUNEL assays. Immunoblot results revealed the upregulation of proapoptotic Bax, cleaved caspase 9 along with Bcl-2, MDM2, Cox-2 and P-Gly down regulation after 24 h drug treatments. Moreover, phospho studies further construed the rationale behind the apoptosis and deduced the inactivation of p38 and NF-κB role in inducing apoptosis in drug treated cells. The efficacy of drug combinations in inactivating p38 was evaluated through drug potential studies. Further, BI-PAC treatments showed inhibition of p38 mediated senescence in tamoxifen resistant cells. Overall, our observations provide a new therapeutic combination that sensitizes tamoxifen resistant cells to apoptosis by specifically targeting p38 signaling and its downstream molecules and subsequently reduces extracellular VEGF levels

Thymoquinone restores radiation-induced TGF-β expression and abrogates EMT in chemoradiotherapy of breast cancer cells

Radiotherapy remains a prime approach to adjuvant therapies in patients with early and advanced breast cancer. In spite of therapeutic success, metastatic progression in patients undergoing therapy, limits its application. However, effective therapeutic strategies to understand the cellular and molecular machinery in inhibiting radiation‐induced metastatic progression, which is poorly understood so far, need to be strengthened. Ionizing radiation was known to prompt cancer cell's metastatic ability by eliciting Transforming Growth Factor‐beta (TGF‐β), a key regulator in epithelial–mesenchymal transdifferentiation and radio‐resistance. In this viewpoint, we employed thymoquinone as a radiosensitizer to investigate its migration and invasion reversal abilities in irradiated breast cancer cell lines by assessing their respective attributes. The role of metastasis regulatory molecules like TGF‐β, E‐cadherin and integrin αV and its downstream molecules were determined using RT‐PCR, western blotting, immunofluorescence and extracellular TGF‐β levels affirmed through ELISA assays. These studies affirmed the TGF‐β restoring ability of thymoquinone in radiation‐driven migration and invasion. Also, results demonstrated that the epithelial markers E‐cadherin and cytokeratin 19 were downregulated whereas mesenchymal markers like integrin αV, MMP9 and MMP2 were upregulated by irradiation treatment; however thymoquinone pre‐sensitization has reverted the expression of these proteins back to control proteins expression. Here, paclitaxel was chosen as an apoptosis inducer in TGF‐β restored cells and confirmed its cytotoxic effects in radiation alone and thymoquinone sensitized irradiated cells. We conclude that this therapeutic modality is effective in preventing radiation‐induced epithelial–mesenchymal transdifferentiation and concomitant induction of apoptosis in breast cancer

Fe₃O₄@zirconium phosphate core–shell nanoparticles for pH-sensitive and magnetically guided drug delivery applications

In the present study, biocompatible and magnetic Fe₃O₄@zirconium phosphate core–shell nanoparticles, comprising a magnetic Fe₃O₄ core and biocompatible zirconium phosphate shell, were prepared for pH-sensitive and magnetically guided drug delivery applications. The formation of the core–shell nanoparticles was affirmed from the XRD and FT-IR studies. TEM analysis revealed proper coverage of the zirconium phosphate shell on the magnetite core, while magnetic studies substantiated the superparamagnetic behavior of the nanoparticles. The suitability of the nanoparticles for intracellular application was validated through the conventional MTT assay, performed on human breast cancer cell lines (MDA-MB-231). The loading-release behavior of the antitumor drug, curcumin, on and from the core–shell nanoparticles respectively were studied by UV-vis spectroscopy, where the drug-loaded particles showed pH-sensitive release profiles. The nanoformulated curcumin was found to exhibit enhanced cytotoxicity and better bioavailability in contrast to native curcumin