Anticancer activity of betulinic acid on MCF-7 tumors in nude mice.

Breast cancer is a major public health problem and the low effectiveness of conventional therapies to achieve long-term survival results in increased mortality associated with advanced breast cancers. Betulinic acid (BA) is a pentacyclic triterpene which can be isolated from number of plants grown in the tropics. It exhibits cytotoxic activity against variety of cancer cell lines. In the present study, the in vitro cytotoxic activity and in vivo antitumor activity of BA was evaluated in athymic nude mice bearing MCF-7 breast adenocarcinoma xenografts. In vitro cytotoxic activity of BA on MCF-7 cells was studied using the MTT assay and BA was cytotoxic towards MCF-7 cells with IC50 value of 13.5 microg/mL. The antitumor activity of BA was studied at concentrations of 50 and 100 mg/kg body weight in mice injected with MCF-7 cells. BA treatment delayed tumor formation and statistically significant reduction (P < 0.0001) of 52 and 77% in the tumor size at concentrations of 50 and 100 mg, respectively was observed. Histopathological analysis of tumors revealed decreased angiogenesis, proliferation and invasion in BA treated animals. This is one of the first studies demonstrating the in vivo antitumor activity of BA on MCF-7 breast cancer tumors in nude mice. The antitumor effect of BA can further be enhanced by use of combination therapy and novel drug delivery systems, thus making it a promising candidate for management of breast cancer patients.

Radioiodide uptake and sodium iodide symporter expression in breast carcinoma.

Breast cancer is a common malignancy in women all over the world and novel therapeutic approaches are required for the treatment of patients who become refractory to conventional therapies. Thyroid cancer is being treated successfully with radioiodine since many years. The iodide is transported inside the thyroid epithelial cell via sodium iodide symporter (NIS) which is a trans-membrane protein. The present study was aimed to explore the uptake of radioiodide (RAI) and the expression of NIS in breast tissues of invasive ductal carcinoma patients. Breast tissues from tumor region (Tu-Br) as well as corresponding normal region (N-Br) were collected from patients of invasive ductal carcinoma. In vitro RAI uptake, its efflux and NIS expression were studied. The uptake of RAI (1.98+/-1.75 x 10(5) cpm/g) in Tu-Br was significantly higher as compared to that observed in N-Br (0.31+/-0.27 x 10(5) cpm/g) and fast efflux was observed in the tissue samples. NIS gene expression was positive in 41.66% (10/24) samples of Tu-Br. None of the N-Br samples expressed NIS gene. In 14 samples of Tu-Br, RAI uptake as well as NIS expression was studied. In 50% of these Tu-Br samples RAI uptake as well as of NIS gene expression was positive. The results indicate that RAI uptake is significantly higher in breast tumor tissues as compared to their normal counterpart and in future radioiodine may be an important agent for treatment of breast cancer.

Betulinic acid induces apoptosis in human chronic myelogenous leukemia (CML) cell line K-562 without altering the levels of Bcr-Abl.

Betulinic acid (BA), a plant derived triterpenoid, isolated from various sources shows cytotoxicity in cell lines of melanoma, neuroectodermal and malignant brain tumors. Chronic myelogenous leukemia (CML) is characterized by Philadelphia chromosome (Bcr-Abl), a molecular abnormality leading to the intrinsic tyrosine kinase activity that provides growth and survival advantage to the cells. Present study describes the cytotoxicity of BA on human CML cell line K-562, positive for Bcr-Abl. The decrease in the viability of K-562 cells treated with BA at different concentrations and time intervals was assessed using MTT assay. Cell death induced by BA was determined to be apoptotic as measured by FACS analysis of PI stained nuclei, PS externalization by Annexin-V fluorescence and characteristic DNA fragmentation. DiOC6(3) fluorescent probe determined alterations in the mitochondrial membrane potential (MMP). RT-PCR confirmed the expression levels of Bcr-Abl in controls and K-562 cells treated with BA. The rapid loss of MMP of K-562 cells upon treatment with BA shows the direct activation of apoptosis at the level of mitochondria, overcoming the resistance of the high levels of expression of Bcr-Abl.

Localization of radiolabeled monoclonal antibodies in thyroid tumor xenografts.

Monoclonal antibodies to human thyroglobulin were produced using the hybridoma technique. Two monoclonal antibodies D5I and F9I were radiolabeled with 125I and used for radioimmunolocalization studies in an immunosuppressed animal model bearing xenografts of human thyroid tumor tissue. Biodistribution studies were carried out at various time intervals post-injection. Maximum tumor uptake was obtained at 72 hr after administration of the antibodies. The absolute tumor uptake (ATU) expressed as percentage of injected dose per gram of tissue (% ID/g) was 15.49 +/- 2.47, 4.51 +/- 0.69 and 2.50 +/- 0.41 for D5I, F9I and control Igs respectively. The tumor to blood ratios (T/B) obtained were 3.01 +/- 0.43 for D5I, 0.98+/-0.2 for F9I and 0.47 +/- 0.12 for control Igs. ATU as well as T/B ratio obtained with D5I was significantly higher as compared to F9I and control Igs. The results indicated the potential application of radiolabeled monoclonal antibodies to human thyroglobulin for tumor targetting in patients of differentiated thyroid carcinoma, particularly those metastases which did not concentrate radioiodine.

BRAF V600E mutation in papillary thyroid carcinoma: significant association with node metastases and extra thyroidal invasion.

B-Raf (BRAF) is the strongest activator in the downstream of MAP kinase signaling. The somatic point mutation of BRAF gene (V600E) is the most common and specific event in papillary thyroid carcinoma (PTC). However, its prevalence is variable among different studies and its association with clinico-pathological features is controversial. This study tests the prevalence of BRAF (V600E) mutation in thyroid cancer patients in Indian subcontinental population. We analyzed 140 thyroid tumor specimens for BRAF gene mutation at codon 600 using mutant-allele-specific amplification, single-strand conformation polymorphism, Mutector assay, and DNA sequencing of the PCR-amplified exon 15. BRAF mutation at codon 600 was detected in 46 of 86 PTC patients (53.4%) from Indian subcontinental cohort. Frequency of mutation varied across the subtypes of PTCs. BRAF (V600E) mutation was more common in the conventional PTC (38 out of 62; 61%) than in the follicular variant of PTC (2 out of 17; 11.7%). None of the 8 follicular thyroid adenomas, 14 follicular thyroid carcinomas, 16 medullary thyroid carcinomas, and 16 benign hyperplasia patients showed any exon 15 mutation. We found significant correlation between BRAF mutation status and extra-thyroidal invasion, lymph node metastasis, and tumor stage. However no correlation was observed with gender, age, and tumor size of the patients. Thus our findings suggest that BRAF (V600E) is a prevalent genetic alteration in adult sporadic PTCs in Indian cohort and it may be responsible for the progression of classic variant of PTC to metastatic and poorly differentiated subtype and likely to have significant impact on its diagnostic and prognostic management.

A single-chain antibody fragment against human thyroglobulin: construction and evaluation of immunoreactivity.

Smaller recombinant antibody fragments are at the forefront of in vivo diagnosis and therapy. These units possess better distribution and faster clearance than larger molecules. Among these, single chain antibody fragments (scFv) are emerging as credible alternatives. These proteins are shown to have same specificities and affinities for their antigens as the parental monoclonal antibody (MAb). We have attempted to produce scFv against human thyroglobulin (H-Tg) using anti-Tg secreting hybridoma cells and PCR-based cloning approach. Hybridoma secreting anti-Tg MAb B10IV was established. cDNA was prepared from hybridoma cells. The V(H) and V(L) genes were amplified and cloned. The gene sequences were submitted to Genebank database (accession nos. AJ508533 and AM072962, respectively.) V(L) and V(H) genes were then linked together with a linker peptide and successfully cloned in pET28a and expressed as His-tag fusion protein in expression host BL21 (DE3). The scFv protein from IPTG-induced cells was purified under native conditions by immobilized metal affinity chromatography on a Ni-NTA agarose column. The yield expressed in Escherichia coli was approximately 8 mg/L. ScFv could be labeled with (125)I and its immunoreactivity evaluated in radioassays. Although scFv demonstrated specific binding to H-Tg, the immunoreactivity was low (10.3%) compared to the parental MAb B10IV, which showed immunoreactivity of 37.27%. Inhibition radioassays exhibited that scFv and MAb interact with the same epitope on the target antigen, indicating its specificity.