Novel 1,4-dihydropyridine induces apoptosis in human cancer cells through overexpression of Sirtuin1.

1,4-Dihydropyridines (1,4-DHPs) are important as a class of heterocyclic compounds that exhibit wide range of biological actions. Many of its derivatives are already characterized as medicinally important drugs and used worldwide. In this study, we have screened some novel Hantzsch 1,4-DHP compounds using both in silico (QSAR and Pharmacophore) and in vitro (cytotoxic screening). 1,4-DHP showed selective cytotoxicity against five human cancerous cell lines; A375, A549, HeLa, HepG2 and SH-SY5Y but limited effect towards normal skin keratinocyte (HaCaT), lung fibroblast (WL-38) and healthy peripheral blood mononuclear cells. In A375 and HepG2 cells, one of the 1,4-DHP derivative (DHP-8) was found to inhibit cell proliferation, and simultaneously increased the apoptotic population as well as mitochondrial membrane depolarization. Furthermore, the mitochondrial signal was triggered with the activation of cleaved Caspase9, Caspase3 and PARP. The treatment with DHP-8 also increased the expression level of SIRT1, subsequently decreasing the level of pAKTser473 and survivin. Reduced pAKTser473 expression led to decrease the phosphorylated inactive form of GSK3ßser9 and as a result, proteasomal degradation of Mcl-1 occurred in both the cell lines. Here, we suggest that the apoptotic effect of DHP-8 in A375 and HepG2 cells was mediated by AKT and survivin pathways through SIRT1 activation. The involvement of DHP-8 in SIRT1 activation was further verified by co-treatment of nicotinamide with DHP-8 in both A375 and HepG2 cells. Overall, this study emphasizes the possible potential and therapeutic role of DHP-8 in skin and liver cancer.

Determinants of lymph node status in women with breast cancer: A hospital based study from eastern India.

BACKGROUND & OBJECTIVES: Number of metastatic lymph nodes has a strong prognostic value in the course of breast cancer treatment, morbidity and mortality. This study was undertaken to determine the association between axillary lymph node metastasis and several variables such as age, tumour size, grade, lymphovascular invasion, oestrogen and progesterone receptor expression and HER2/neu status in patients with breast cancer. METHODS: In this study 426 (with complete information on study variables) patients with breast cancer on treatment during March 2010 to December 2013, were analyzed. TNM (tumour node matastasis) staging was evaluated. The histological grading of tumours was done according to modified Bloom-Richardson Grading System. The immunophenotype of the tumour was determined as the expression of oestrogen (ER) and progesterone (PR) receptors and Her0 2/neu status. Univariate and multivariate analyses were carried out to determine the independent predictors of metastatic lymph node. RESULTS: Among the studied patients, 44.36 per cent (189 of 426) of the patients had nodal metastases. t0 umour histology, tumour grade, size and lympho-vascular invasion were related with node positivity. On univariate analysis, age, menopause, hormone receptor status did not relate with the node metastasis. Age, tumour grade, tumour size, lympho-vascular invasion and HER2/neu expression was likely to be associated with the number of lymph node metastasis. INTERPRETATION & CONCLUSIONS: The lymph node status was associated with clinical stage, tumour grade, tumour histology and HER2/neu status. t0 hese factors may be used for better management of such patients.

Absence of 185delAG and 6174delT Mutations among Breast Cancer Patients of Eastern India.

BACKGROUND: The incidence of breast cancer in India is on the rise and is rapidly becoming the number one cancer in females, pushing the cervical cancer to the second position. Most of the predisposition to hereditary breast and ovarian cancer has been attributed to inherited defects in two tumor suppressor genes BRCA1 and BRCA2. Alterations in these genes have been reported in different populations, some of which are population- specific mutations showing founder effects. Two specific mutations in the BRCA1 (185delAG) and BRCA2 (6174delT) genes have been reported to be of high prevalence in different populations. The aim of this study was to estimate the carrier frequency of 185delAG and 6174delT mutations in eastern Indian breast cancer patients. MATERIALS AND METHODS: We selected 231 histologically confirmed breast cancer patients from our tertiary cancer care center in eastern India. Family history was obtained by interview or a self-reported questionnaire. The presence of the mutation was investigated by allele specific duplex/multiplex-PCR on genomic DNA extracted from peripheral blood. RESULTS: A total of 231 patients (age range: 26-77 years), 130 with a family history and 101 without were screened. The two founder mutations 185delAG in BRCA1 and 6174delT in BRCA2 were not found in any of the subjects. This was confirmed by molecular analysis. CONCLUSIONS: Our findings suggest that these BRCA mutations may not have a strong recurrent effect on breast cancer among the eastern Indian population. The contribution of these founder mutations to breast cancer incidence is probably low and could be limited to specific subgroups. This may be particularly useful in establishing further pre-screening strategies.

Coexistence of rare variant HbD Punjab [α2ß2(121(Glu→Gln))] and alpha 3.7 kb deletion in a young boy of Hindu family in West Bengal, India.

HbD Punjab is a variant of hemoglobin which occurs as a result of mutation in codon 121 (GAA>CAA) of the ß-globin gene, which replaces glutamic acid with glutamine (Glu→Gln). The heterozygous state of HbD does not produce any clinical or hematological symptoms, although its association with HbS and thalassemia produces clinically significant but less severe conditions. The homozygous state produces mild hemolytic anemia and mild to moderate splenomegaly. Alpha-thalassemia is characterized by reduction or absence of the α-globin chains due to deletional or non-deletional mutations of α-globin genes located on chromosome 16. The present study describes a Hindu family where both HbD Punjab and alpha 3.7 kb deletion are present among the members in the heterozygous and double heterozygous state. Comparison of clinical and hematological parameters between the heterozygous and double heterozygous state of HbD and the alpha 3.7 kb deletion is also discussed here. According to our study, the prevalence rate of HbD Punjab is very low, i.e. 0.06%.

Fannin-Lubbock-I [α2ß2¹¹9(GLY>ASP)], a rare mutation in the beta-globin gene, has been detected for the first time in a Hindu Brahmin family in West Bengal, India.

This study aims to describe the hemoglobin Fannin-Lubbock-I, which has a rare mutation substituting the amino acid glycine with aspartic acid at codon 119 of the ß-globin chain. A Bengalee Hindu Brahmin family from Kolkata in West Bengal was the focus of this study. Molecular analysis using ARMS-PCR and direct DNA sequencing revealed the presence of a GGC > GAC mutation in codon 119 of the ß-globin gene in a heterozygote state in three women of the same family. This is the first report of the hemoglobin Fannin-Lubbock-I from India. Our results will help to identify this mutation, which is relatively infrequent in our population.

Descriptive profile of ß-thalassemia mutations in West Bengal population: a hospital-based study.

The present study was based in a hospital at which 660 individuals have been screened for thalassemia in the past 4 years. The main purposes of the study were to identify different types of beta mutations prevailing among these patients, and to establish a genotype-phenotypic correlation. Complete blood count, high-performance liquid chromatography, and amplification refractory mutation system-based polymerase chain reaction were performed on peripheral blood samples to detect beta mutations. Of the 660 subjects studied, 380 (57.6 %) were male and 280 (42.4 %) were female. These included 258 (39.09 %) normal individuals, 176 (26.67 %) ß-thalassemia carriers, 44 (6.67 %) ß-thalassemia major, 6 (0.91 %) cases of sickle ß-thalassemia, 6 (0.91 %) carriers of sickle cell anemia, 102 (15.45 %) Hb Eß-thalassemia, 42 (6.36 %) HbE carriers, 16 (2.42 %) HbE homozygous, and 10 (1.52 %) carriers of other mutations. Genotypic study of beta mutations revealed the prevalence of IVS1-5 mutation among the studied beta carriers to be 46.6 %, and codon 26 (G>A) mutation to be 31.54 %. Other prevailing mutations among the screened individuals include codon 30 (7.53 %), codon 15 (5.01 %), codon 41/42 (3.58 %), and codon 8/9 (1.07 %). Genotype-phenotype correlation revealed that the phenotype of the above-mentioned mutations is associated with mild, moderate, and severe forms of thalassemia.

Erratum to: Detection of a novel mutation in exon 20 of the BRCA1 gene.

[This corrects the article DOI: 10.2478/s11658-013-0110-3.].

Detection of a novel mutation in exon 20 of the BRCA1 gene.

Hereditary breast cancer constitutes 5-10% of all breast cancer cases. Inherited mutations in the BRCA1 and BRCA2 tumor-suppressor genes account for the majority of hereditary breast cancer cases. The BRCA1 C-terminal region (BRCT) has a functional duplicated globular domain, which helps with DNA damage repair and cell cycle checkpoint protein control. More than 100 distinct BRCA1 missense variants with structural and functional effects have been documented within the BRCT domain. Interpreting the results of mutation screening of tumor-suppressor genes that can have high-risk susceptibility mutations is increasingly important in clinical practice. This study includes a novel mutation, p.His1746 Pro (c.5237A>C), which was found in BRCA1 exon 20 of a breast cancer patient. In silico analysis suggests that this mutation could alter the stability and orientation of the BRCT domain and the differential binding of the BACH1 substrate.

Frequency of 5382insC mutation of BRCA1 gene among breast cancer patients: an experience from Eastern India.

The incidence of breast cancer in India is on the rise and is rapidly becoming the number one cancer in females pushing the cervical cancer to the second position. The mutations in two breast cancer susceptibility genes, BRCA1 and BRCA2, are frequently associated with familial breast cancer. The main objective of the study was to determine the frequency of the mutation 5382insC in BRCA1 of eastern Indian breast cancer patients and also study the hormonal receptor status and histopathology of the patients. Altogether 92 patients affected with breast cancer were included in this study. ARMS-PCR based amplification was used to detect the presence of mutation. The mutations were considered only after pedigree analysis. Out of 92 patients (age range: 20-77 years) with family history (57 individuals) and without family history (35 individuals) were screened. Fifty controls have been systematically investigated. Seven patients and two family members were found to be carriers of 5382insC mutation in BRCA1 gene. We have found 42.64 % ER(-)/PR(-) cancer and 20.58 % triple negative cancer. Invasive ductal carcinoma is the most common histology among the investigated individuals. The presented data confirm a noticeable contribution of BRCA1 5382insC mutation in BC development in Eastern India, which may justify an extended BRCA1 5382insC testing within this patient population. We found HER-2/neu negativity and BRCA1 positivity associated with familial breast cancer. From the hospital's patient history, it was revealed that the age of menarche plays an important role in development of breast cancer.

Incidence of the Hb E [ß26(B8)Glu→Lys, GAG>AAG] variant in Totos, one of the smallest primitive tribes in the world.

Toto is one of the smallest tribes in the world. This primitive sub Himalayan, endogamous tribe lives in a small, isolated village called Totopara in the Jalpaiguri district of West Bengal in India. The tribal communities of West Bengal are vulnerable to various genetic disorders such as ß-thalassemia (ß-thal). We have studied 443 Totos to define their Hb E [ß26(B8)Glu→Lys, GAG>AAG] status. Awareness and screening camps have been organized in various parts of Totopara during the last 2 years. We collected 3 mL peripheral blood from each individual aseptically on which to use the naked eye single tube red cell osmotic fragility test (NESTROFT); complete hemogram and high performance liquid chromatography (HPLC) were done to detect their carrier status. The Hb E variant had been found to be prevalent among the Totos. To confirm the codon 26 (GAG>AAG) mutation in the ß-globin gene, amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) was performed. Restriction fragment length polymorphism (RFLP)-PCR was carried out with 44 Hb E alleles to construct the haplotype(s) of the Totos. Our extensive studies have revealed that 49.21% of Totos are Hb E heterozygotes and 19.19% Totos are Hb E homozygotes. The most prevalent haplotype linked with the codon 26 mutation in the Totos is [+ - - - - -] (HincII 5'ϵ, HindIII (G)γ, HindIII (A)γ, HincII 5'ψß, HincII 3'ψß and HinfI 3'ß). Consanguineous marriages have resulted in a significant increase of the percentages of heterozygotes and homozygotes of Hb E in the Totos. Genetic counseling is essential and important to prevent the spread of this mutation and hence to save them from having any kind of clinically significant hemoglobinopathy in the future.

Deletional mutations of dystrophin gene and carrier detection in eastern India.

OBJECTIVE: To determine the pattern of deletions of the dystrophin gene, the major class of mutations among the Duchenne and Becker muscular dystrophy patients of eastern India and to analyze the carrier frequency of the female members of the proband's family. METHODS: Deletional mutations occurring in patients have been characterized by multiplex polymerase chain reaction. Carrier state of mothers and sisters of probands were analyzed by either of two methods: 1) typing polymorphic short tandem repeat markers in or around the regions of deletion, by radioactive polymerase chain reaction and 2) quantitative real time amplification of the region of deletion. RESULTS: Deletions were detected in 67 (62.04%) out of 108 male patients, about 76.12% of these being localized in the central hot spot region of the gene, i.e., between exon 42 to exon 53 and 17.91% at the proximal hot spot i.e., between exon 1 to exon 20. In the present study were found 43 types of deletions, out of which 25 (58%) were new deletions, which were not described earlier among the Indian patients. Distribution pattern of deletions in different hot spot regions has been compared with that of other countries and statistical analysis reveals significant difference between countries (p<0.001). Correlation of the pattern of deletion with clinical phenotype of patients has been discussed. Interesting case of germline mosaicism and its implications in counseling has also been discussed. CONCLUSION: About half the mothers of affected probands were not carriers of the deletion, underscoring the need to use real time techniques for carrier detection.

Analysis of dystrophin gene deletions by multiplex PCR in eastern India.

The most common genetic neuromuscular disease of childhood, Duchenne and Becker muscular dystrophy (DMD/BMD) is caused by deletion, duplication or point mutation of the dystrophin gene located at Xp 21.2. In the present study DNA from seventy unrelated patients clinically diagnosed as having DMD/BMD referred from different parts of West Bengal, a few other states and Bangladesh are analyzed using the multiplex polymerase chain reaction (m-PCR) to screen for exon deletions and its distribution within the dystrophin gene. Out of seventy patients forty six (63%) showed large intragenic deletion in the dystrophin gene. About 79% of these deletions are located in the hot spot region i.e, between exon 42 to 53. This is the first report of frequency and distribution of deletion in dystrophin gene in eastern Indian DMD/BMD population.

Profile of beta-thalassemia in eastern India and its prenatal diagnosis.

OBJECTIVE: To control the birth of thalassemic children in India. METHODS: Mutations present in the population of eastern India and in carrier parents seeking prenatal diagnosis were detected by the PCR-based technique of ARMS (amplification refractory mutation system) or gap-PCR. To screen for maternal tissue contamination in CVS, haplotypes associated with the beta-globin gene clusters were constructed using six polymorphic restriction sites. Prenatal diagnosis was accomplished by checking presence of parental mutation in the DNA from chorionic villus sampling (CVS) collected at 8 to 10 weeks' gestation by appropriate technique. RESULTS: Six hundred and fifty (650) unrelated beta-thalassemia chromosomes were screened for 11 common mutations to characterize the mutation distribution in this population. Starting from early 2000, 63 families from different parts of West Bengal and from surrounding areas have been offered prenatal counseling for beta-thalassemia. CONCLUSION: The population of this region is conscious and willing to accept prenatal diagnosis as a means of control of thalassemia.