Inhibition of AKT promotes FOXO3a-dependent apoptosis in prostate cancer.

Growth factor-induced activation of protein kinase-B (PKB), also known as AKT, induces pro-survival signaling and inhibits activation of pro-apoptotic signaling molecules including the Forkhead box O-3a (FOXO3a) transcription factor and caspase in transformed prostate cells in vitro. Earlier we reported that Withaferin-A (WA), a small herbal molecule, induces pro-apoptotic response-4 (Par-4) mediated apoptosis in castration-resistant prostate cancer (CRPC) cells. In the present study, we demonstrate that inhibition of AKT facilitates nuclear shuttling of FOXO3a where it regulates Par-4 transcription in CRPC cells. FOXO3a is upstream of Par-4 signaling, which is required for induction of apoptosis in CRPC cells. Promoter bashing studies and Ch-IP analysis confirm a direct interaction of FOXO3a and Par-4; a sequential deletion of FOXO3a-binding sites in the Par-4 promoter fails to induce Par-4 activation. To confirm these observations, we either overexpressed AKT or silenced FOXO3a activation in CRPC cells. Both methods inhibit Par-4 function and apoptosis is significantly compromised. In xenograft tumors derived from AKT-overexpressed CRPC cells, FOXO3a and Par-4 expression is downregulated, leading to aggressive tumor growth. Oral administration of WA to mice with xenograft tumors restores FOXO3a-mediated Par-4 functions and results in inhibited tumor growth. Finally, an inverse correlation of nuclear localization of AKT expression corresponds to cytoplasmic Par-4 localization in human prostate tissue array. Our studies suggest that Par-4 is one of the key transcriptional targets of FOXO3a, and Par-4 activation is required for induction of apoptosis in CRPC cells. Activation of FOXO3a appears to be an attractive target for the treatment of CRPC and molecules such as WA can be explored further for the treatment of CRPC.

The pro-apoptotic role of autophagy in breast cancer.

BACKGROUND: Autophagy is a catabolic process that has a vital role in cancer progression and treatment. Current chemotherapeutic agents, which target autophagy, result in growth inhibition in many cancer types. In this study, we examined the role of autophagy in breast cancer (BCa) patients as well as BCa cell lines. METHODS: Tissue microarray was used to detect the expression of an autophagy marker, LC3B in BCa patients (normal/hyperplasia=8; grade-I=15, grade-II=84, and grade-III=27) and BCa cell lines. To modulate the activation of autophagy, we used novel herbal compound nimocinol acetate (NA) in BCa cell lines and the anticancer activity was measured by phenotypic and molecular analysis. RESULTS: LC3B is highly expressed in tumours as compared with normal tissues. Activation of LC3B in NA-treated BCa (MCF-7 and MDA-MB-231) cells was evident as compared with other autophagy makers. Further, our results confirmed that NA-transcriptionally regulates LC3B (as confirmed by mRNA levels and reporter assay), which resulted in the formation of acidic autophagy vesicles and autolysosomes in BCa cells. Nimocinol acetate inhibited mTOR-mediated pro-survival signalling that resulted in inhibition of growth in BCa cells without affecting normal breast epithelial cells. Downregulation of LC3B expression by siRNA significantly inhibited the anticancer effects of NA in BCa cells. CONCLUSIONS: Together, our results suggest that LC3B is highly expressed in BCa tissues and increasing the threshold of LC3B activation dictates the pro-apoptotic function, which in turn, suppresses the growth of BCa cells. Nimocinol acetate could be a potential agent for treatment of BCa.

Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells.

BACKGROUND: Breast cancer stem cells (BCSCs) are characterized by high aldehyde dehydrogenase (ALDH) enzyme activity and are refractory to current treatment modalities, show a higher risk for metastasis, and influence the epithelial to mesenchymal transition (EMT), leading to a shorter time to recurrence and death. In this study, we focused on examination of the mechanism of action of a small herbal molecule, psoralidin (Pso) that has been shown to effectively suppress the growth of BSCSs and breast cancer cells (BCCs), in breast cancer (BC) models. METHODS: ALDH(-) and ALDH(+) BCCs were isolated from MDA-MB-231 cells, and the anticancer effects of Pso were measured using cell viability, apoptosis, colony formation, invasion, migration, mammosphere formation, immunofluorescence, and western blot analysis. RESULTS: Psoralidin significantly downregulated NOTCH1 signaling, and this downregulation resulted in growth inhibition and induction of apoptosis in both ALDH(-) and ALDH(+) cells. Molecularly, Pso inhibited NOTCH1 signaling, which facilitated inhibition of EMT markers (ß-catenin and vimentin) and upregulated E-cadherin expression, resulting in reduced migration and invasion of both ALDH(-) and ALDH(+) cells. CONCLUSION: Together, our results suggest that inhibition of NOTCH1 by Pso resulted in growth arrest and inhibition of EMT in BCSCs and BCCs. Psoralidin appears to be a novel agent that targets both BCSCs and BCCs.

Distribution of alleles of 12 STR loci in Tamil population (south India).

Distribution of allele frequencies for 12 STR loci (CSFIPO, TPOX, THO1, F13A01, FESFPS, vWA, D16S539, D7S820, D13S317, HPRTB, F13B and LPL) has been studied for the first time in unrelated Tamil (south India) population.

Mayaro virus disease: an emerging mosquito-borne zoonosis in tropical South America.

This report describes the clinical, laboratory, and epidemiological findings on 27 cases of Mayaro virus (MV) disease, an emerging mosquito-borne viral illness that is endemic in rural areas of tropical South America. MV disease is a nonfatal, dengue-like illness characterized by fever, chills, headache, eye pain, generalized myalgia, arthralgia, diarrhea, vomiting, and rash of 3-5 days' duration. Severe joint pain is a prominent feature of this illness; the arthralgia sometimes persists for months and can be quite incapacitating. Cases of two visitors from the United States, who developed MV disease during visits to eastern Peru, are reported. MV disease and dengue are difficult to differentiate clinically.

Multilocus DNA profiling using the probe 33.6 in the Tamil Nadu population of South India.

Using the multilocus minisatellite probe 33.6 in combination with the HinfI restriction enzyme, the extent of genetic variation detected by DNA fingerprinting was estimated in 102 unrelated individuals of the Dravidian Hindu population of Tamil Nadu, South India. In this first study of its kind on an anthropologically defined Indian population, DNA fragments of size >2.5 kb could be reliably scored. Results indicate that the Tamilian Hindus show an average number of bands per individual somewhat smaller (15.69 for fragments of size >2.5 kb) than that in other Caucasian populations. For comparable molecular weight of fragment sizes, the Tamilian Hindus show a lower level of band sharing probabilities between unrelated individuals compared with other Caucasians. Nevertheless, the probe 33.6 offers a high level of individualization of DNA fingerprints for unrelated individuals in Tamil Nadu. Computations on expected band sharing frequencies between various biological relatives and expected DNA fingerprint identity indicate that this multilocus minisatellite probe can be efficiently used in resolving forensic identification and parentage testing cases in this South Indian population in spite of its high level of inbreeding. Am. J. Hum. Biol. 10:87-93, 1998. © 1998 Wiley-Liss, Inc.

Hidden monosomy 7 in acute myeloid leukemia and myelodysplastic syndrome detected by interphase fluorescence in situ hybridization.

Fifty patients [25 acute myeloid leukemia (AML) and 25 myelodysplastic syndrome (MDS)], without monosomy 7 according to conventional cytogenetics, were re-examined by fluorescence in situ hybridization (FISH). Eleven (44.0%) patients with AML and nine (36.0%) with MDS showed hidden monosomy 7. Two samples who had both monosomy 7 and iso chromosome 17 were analyzed by dual color FISH to identify their clonal origin, and showed that these two abnormalities can occur together or independently. Only one of 16 MDS patients without monosomy 7 transformed into AML whereas four of eight MDS patients with the hidden monosomy 7 transformed into AML, suggesting patients with this abnormality are more likely to undergo transformation to AML.

Allele frequency distribution for the variable number of tandem repeat locus D10S28 in Tamil Nadu (south India) population.

Allele frequencies were determined in unrelated individuals of Tamil speaking population from the Madras City (Tamil Nadu, South India) area for the polymorphic DNA locus D10S28 using the probe TBQ7. Membranes hybridized with the probe YNH24 were subjected to deprobing and were subsequently hybridized with random priming - labeled, purified inserts of TBQ7. The sizes of the fragments were grouped to 100 bp as well as to arbitrary fixed bins (Federal Bureau of Investigation / Royal Canadian Mounted Police). There were 14 bins in the latter with the most common bin being 11 (1789-1924 bp) with a frequency of 9.8%. We observed a heterozygosity of 92% comparable to Caucasian populations. The data presented here can be used as the basis for utilizing this variable number of tandem repeats (TNTR) DNA marker for paternity determinations and forensic investigations.

Distribution of HLA antigens in Sadhu Chetty of Tamil Nadu, south India.

Blood samples from 101 individuals of Sadhu Chetty community domiciled in and around Madras (capital City of Tamil Nadu State, South India) were examined for HLA-A, B, C, DR and DQ antigen profiles. Phenotype, gene and haplotype frequencies were calculated and compared with the literature. Increased frequencies of HLA-B16 antigen and of the haplotype A2, B16 were characteristic. This study indicates the distinctiveness of the Sadhu Chetty population and highlights the importance of determining HLA frequencies in endogamous groups of India.

DNA profiling of the VNTR locus D2S44 in the population of Madras City and in the tribal Kotas of the Nilgiri Hills, south India.

Allele frequency distribution of the VNTR locus D2S44 was studied in Tamil Nadu (South India) population. Randomly chosen individuals (Tamils of the plains, Madras City; N = 142) were tested for HaeIII-generated polymorphism detectable by probe YNH24, and the allele sizes and frequencies were determined. Heterozygosity (93.6%) observed in the Tamils is comparable to that of other populations; the size and frequency distribution of alleles, however, vary significantly. The most prevalent allele, which ranges from 1.2 to 1.9 kb, appears to be unique to the Tamil Nadu population. No mutation was observed for the D2S44 locus in family material made up by 54 subjects (N = 54) including 37 offspring. DNA polymorphism at D2S44 locus was also studied in the endogamous Kota tribe of the Nilgiri Hills, South India, using enzyme HaeIII and probe YNH24. The Kota group (N = 48) is characterized by a very high frequency (32.3%) of the bin 1197-1352 bp. The DNA profile of the Kotas shows distinct differences from that of the urban population in the plains of Tamil Nadu, South India (Tamils of Madras City). The results have also been compared with the literature available on other world populations. The outcome indicates the uniqueness of the tribal Kotas and warrants the importance of DNA profiling in other tribal, caste/endogamous groups of India. This report incidentally represents the first comprehensive DNA profiling data for the locus D2S44 from India fulfilling the requirement for forensic and other applications.

Distribution of HLA (class I and class II) antigens in the native Dravidian Hindus of Tamil Nadu, south India.

HLA - A, B, C, DR, DQ antigen profile of South Indian Tamil-speaking Hindus of Dravidian descent was studied. Phenotype, gene and haplotype frequencies were calculated and compared with the literature. There was a complete lack of A23, A25 and A32 antigens in the sample presently monitored. Except for minor differences (higher incidence of Cw6 and DR10 antigens), the Dravidian Hindus show similarity to North Indio-Aryan and other Hindu samples. The haplotypes A1, B17; A2, B5; A2, B51; A1, DR7; B12, DR7; B13, DR2; B17, DR7; DR2, DQ1; DR3, DQ2; DR4, DQ3; DR5, DQ3; DR7, DQ2; DR11, DQ3; show significant positive linkage disequilibrium whereas A1, DR2; DR2, DQ2; DR7, DQ1 were significant for negative linkage disequilibrium in the Dravidian Hindus.

Red cell enzymes and serum protein polymorphisms in three population groups of south India.

Blood and serum samples from random individuals of three populations in south India, the first being an endogamous group from the Nilgiri hills (Tamil Nadu), the second from the Shevroy hills (Tamil Nadu), and the third from a semi-urban area of Tamil Nadu, were screened for ESD, GLO1 and Hp polymorphisms. The allelic frequencies for these markers have been estimated. High GLO1*1 (0.379) frequency was observed in the tribal Malayalis, in contrast with other Indian population groups.

HLA, ESD, GLOI, C3 and HP polymorphisms and juvenile insulin dependent diabetes mellitus in Tamil Nadu (south India).

Fifty juvenile insulin dependent diabetes mellitus (JIDDM) patients of Tamil Nadu (South India) were typed for HLA-A, -B, -C, -DR, and -DQ, ESD, GLOI, C3 and HP polymorphisms. The frequencies of B8, DR3, DR4, DR53 and DQ2 antigens of the HLA system were significantly higher in the patients than in controls (relative risk, RR = 4.81; 5.14; 3.98; 3.36 and 2.53, respectively). However HLA-DR2, -DR5 and -DQ1, observed less frequently in the patient group, appear to play a role of protection against the disease (RR = 0.32; 0.30 and 0.20 respectively). HLA haplotype analysis demonstrated very high relative risk associated with two hitherto unreported haplotypes namely A3,DR1 and Cw3,DR4 (RR = 27.30 and 20.00, respectively) and also scanty distribution of the haplotypes A1,B17 and DR2,DQ1 (RR = 0.39 and 0.36, respectively) in the patient group. Among other genetic markers tested, GLOI is informative with its phenotype GLOI 2-1 showing positive association with JIDDM (RR = 4.06).

Enzyme-linked immunosorbent assay for the phytotoxin thevetin.

An enzyme-linked immunosorbent assay is reported for monitoring thevetin, an active constituent of the highly poisonous plant Thevetia nerifolia. A thevetin-BSA conjugate was employed as the immunogen and the antibodies raised in rabbits were used for the development of an ELISA. Penicillinase served as the marker enzyme and its conjugation to thevetin by the periodate method is reported for the first time. The present ELISA method could detect 2 ng/ml of thevetin. Cross-reactivity studies with structural analogues and other phytotoxins and drugs of common occurrence in clinical and forensic toxicology established the superiority of the ELISA over the existing analytical methods for determining thevetin in various biospecimens.

Enzyme-linked immunosorbent assay (ELISA) for the determination of the toxic glycoside cleistanthin B.

Cleistanthus collinus is a highly toxic plant frequently implicated in suicidal and homicidal poisoning cases referred to our laboratory. An enzyme-linked immunosorbent assay (ELISA) is reported in this paper for the quantitation of the active principles of C. collinus. This method is highly reproducible and sensitive to detect as low as 2 ng/ml of cleistanthin B, a toxic constituent of C. collinus. The ELISA can be successfully applied for the specific identification of C. collinus poisoning in clinical and forensic toxicology.