Mapping phenotypic performance and novel SNPs for cold tolerance in tomato (Solanum lycopersicum) genotypes through GWAS and population genetics.

The cold stress susceptibility of tomato (Solanum lycopersicum) curtails its cultivation, with significant impact in temperate regions and on cropping seasons. To unravel genomic regions responsible for cold stress resilience, a diverse set of fifty genotypes encompassing cultivated, wild species, and landraces were genotyped using genotyping-by-sequencing. Over two years and six trials employing both early and late sowing, these lines were evaluated. Illumina-based next-generation sequencing produced up to 3 million reads per sample from individually sequenced library pools. The Tassel pipeline yielded 10,802 variants, subsequently filtered to 3,854 SNPs for genome-wide association analysis (GWAS). Employing clustering methods (population structure) via TASSEL, SNPhylo, and Kinship matrix, the fifty genotypes clustered into four distinct gene pools. The GWAS for cold tolerance in tomato integrated key traits including yield. Using six independent phenotypic datasets representing various environments, the study identified 4,517 significant marker-trait associations for cold tolerance traits. Notably, pivotal variations (> 10%) in cold stress tolerance, particularly proline content, were linked to marker-trait associations. Additionally, 5,727 significant marker-trait associations for yield and yield-related traits were unveiled, shedding light on fruit yield and directly associated attributes. The investigation pinpointed 685 candidate genes across all examined traits, including 60 genes associated with biological processes within these genomic regions. Remarkably, 7 out of the 60 genes were directly linked to abiotic stress tolerance, functioning as stress-responsive genes either directly or indirectly. The identified genes, particularly those associated with stress response, could hold the key to enhancing cold tolerance and overall crop productivity in tomato cultivation.

Enhancement of nutraceutical and anti-diabetic potential of fenugreek (Trigonella foenum-graecum). Sprouts with natural elicitors.

Trigonella foenum-graecum has been extensively used for centuries in traditional medicine systems for the cure of health ailments including diabetes. Improving the medicinal attributes of plants through the elicitation strategy is gaining great interest in the recent past. In the current study, an attempt is made to reveal the role and possible mechanism of action of vitamin C elicit phytochemical-rich aqueous extract of 4th day germinated IM6 genotype fenugreek sprouts in the form of lyophilized powder (IM6E) under both in vitro and in vivo conditions. The IM6E demonstrated strong α-glucosidase activity (95.24 %) and moderate α-amylase and invertase inhibition activities under in vitro conditions. The High Performance Thin Layer Chromatography (HPTLC) based analysis demonstrated that IM6E possess significantly higher concentration of phenolic phytochemical quercetin (0.148 %) as compared to diosgenin and trigonelline bioactive anti-diabetic nutraceuticals. In normal rats after loading with glucose and sucrose, the IM6E administration in a dose-dependent manner significantly reduced the post-prandial hyperglycemia, in a similar fashion as the anti-diabetic drug voglibose as evident from the area under curves (AUC) of oral glucose tolerance test (OGTT) and oral sucrose tolerance test (OSTT) tests. The administration of IM6E in streptozotocin (STZ) induced diabetic rats drastically improved the antioxidant activity of plasma in them as determined by Ferric Reducing Ability of Plasma (FRAP) and the effect was found to be dose-dependent. The oral administration of IM6E in diabetic rats normalized almost all the deregulated biochemical markers like liver enzymes, lipids and significantly decreased higher blood glucose levels with increasing insulin levels as compared to diabetic control. The best concentration of IM6E was found to be 300 mg/kg b.w after 21 days of experimentation. The intra-peritoneal glucose tolerance test (IPGTT) in diabetic rats responded very well to IM6E treatment and 100 mg/kg.b.w. behaved almost like the administration of 0.5U insulin/kg bw, and thus indicating the insulinotropic nature of IM6E. Our findings clearly reveal the use of IM6E for diabetes management and at the same it possesses great potential when combined with voglibose to ameliorate diabetes and its associated complications to a greater extent due to synergistic effects as compared to monotherapy. However, more clinical trials need to be performed before recommending IM6E as an anti-diabetic alternative medicine.

Biochemical and proteomic insights revealed selenium priming induced phosphorus stress tolerance in common bean (Phaseolus vulgaris L.).

BACKGROUND: Mineral stress is one of the dominating abiotic stresses, which leads to decrease in crop production. Selenium (Se) seed priming is a recent approach to mitigate the plant's mineral deficiency stress. Although not an essential element, Se has beneficial effects on the plants in terms of growth, quality, yield and plant defense system thus, enhancing plant tolerance to mineral deficiency. METHODS AND RESULTS: The present research was accomplished to find out the effect of Se priming on common bean plant (SFB-1 variety) under phosphorus (P) stress. The seeds were grown invitro on four different MGRL media which are normal MGRL media as control with non-Se primed seeds (Se- P+), non -Se primed seeds grown on P deficient MGRL media (Se- P-), Se primed seeds grown on normal MGRL media (Se+P+) and Se primed seeds grown on P deficient MGRL media (Se+P -). The various morphological and biochemical parameters such as proline content, total sugar content, polyphenols and expression of proteins were analyzed under P stress. The results showed that Se priming has significantly (p ≤ 0.05) affected the morphological as well as biochemical parameters under normal and P stress conditions. The morphological parameters-length, weight, number of nodes and leaves of Se+P+, Se+P- root and shoot tissue showed significant increase as compared to Se-P+, Se-P-. Similarly various biochemical parameters such as total chlorophyll content, proline, total sugar content and polyphenols of Se+P+, Se+P- increased significantly as compared to Se-P+, Se-P-. The differential protein expression in both Se+P+, Se+P- and Se-P+, Se-P- plants were determined using MALDI-MS/MS. The differentially expressed proteins in Se+P+, Se+P- plants were identified as caffeic acid-3-O-methyltransferase (COMT) and SecA protein (a subunit of Protein Translocan transporter), and are found responsible for lignin synthesis in root cell walls and ATP dependent movement of thylakoid proteins across the membranes in shoot respectively. The differential expression of proteins in plant tissues, validated morphological and biochemical responses such as maintaining membrane integrity, enhanced modifications in cellular metabolism, improved polyphenol activities and expression of defensive proteins against mineral deficiency. CONCLUSIONS: The study provided an understanding of Se application as a potential approach increasing tolerance and yield in crop plants against mineral deficiency.

Nutritional Profiling, Phytochemical Composition and Antidiabetic Potential of Taraxacum officinale, an Underutilized Herb.

Taraxacum officinale (T. officinale), a wild vegetable with a number of health claims, has been mostly ignored and unexplored. The study aims to compare the nutritional, phytochemical as well as antidiabetic potential of fresh as well as shade-dried leaves of T. officinale, in order to recommend its best form as a dietary antidiabetic product. The results revealed that as compared to fresh leaves, the shade-dried leaves, in addition to possessing higher levels of carbohydrates, crude protein, crude fat, crude fiber, etc., also contain appreciable amounts of total phenols (5833.12 ± 4.222 mg/100), total flavonoids (188.84 ± 0.019 mg/100 g), ascorbic acid (34.70 ± 0.026 mg/100 g), ß-carotene (3.88 ± 1.473 mg/100 g) and total chlorophyll (239.51 ± 0.015 mg/100 g) antioxidants. The study revealed the presence of medicinally important antidiabetic flavonoid quercetin present in T. officinale leaves. Among the three solvent systems used, the aqueous extract of shade-dried T. officinale leaves comparatively demonstrated potent antidiabetic activity under in vitro conditions in a dose-dependent manner via targeting α-amylase and α-glucosidase, the two potent enzymes of carbohydrate metabolism. Therefore, in addition to being a nutritious herb, the shade-dried leaves of T. officinale have great potential to suppress post-prandial glucose rise and can be better exploited through clinical trials to be used as a dietary intervention for better management of diabetes.

Polyphenolics, antioxidant characterization and DNA barcoding of Kala zeera [Bunium persicum (Boiss.) Fedtsch] through multiple barcode analysis to unravel best barcode combination.

BACKGROUND: Kala zeera [Bunium persicum (Boiss.) Fedtsch] is one of the important spice crops of North Western Himalayas with lot of medicinal and culinary values. In spite of having great importance, this crop is under the threat of extinction due to loss of habitat and lack of awareness. The limited availability of the seeds has ultimately increased the economic value of this spice. The upmarket of Kala zeera leads to its adulteration with other black seeds and cumin seeds. The present investigation was undertaken to evaluate polyphenolics and antioxidant properties of Kala zeera genotypes collected from North Western Himalayas and to develop DNA barcodes that can ensure their purity and can also guide in conservation of selected Kala zeera germplasm lines. METHODS AND RESULTS: Various locations of North Western Himalayas were explored for collecting 31 diverse germplasm lines of Kala zeera. The collected germplasm was maintained at our experimental stations during 2019-2020 and 2020-2021. These genotypes were evaluated for different seed traits and the methanolic extract from Kala zeera seeds was examined for total phenolic content, total flavonoid content, antioxidant activities by DPPH and FRAP. The results revealed significant variation in seed traits, polyphenolic content and antioxidant properties. 100 seed weight ranged from 0.05 to 0.35 g, TPC ranged from 7.5 to 22.56 mg/g, TFC ranged from 0.58 to 4.15 mg/g, antioxidant properties DPPH ranged from 168 to 624.4 µg/ml and FRAP ranged from 0.72 to 6.91 mg/g. Further, three different barcodes (ITS, rbcL and psbA-trnH) were used to reveal the authenticity of selected Kala zeera. MEGA 5 software was used for clustering and the barcodes did clustering based on geographical distribution of Kala zeera germplasm. CONCLUSION: Based on molecular barcoding, best barcode combination was identified that may discriminate the Kala zeera germplasm vis-a-vis can authenticate their purity. Moreover, the identified DNA barcodes will have significant role in studying the evolutionary biology of Bunium species and will be important for designing a strategy to conserve the selected Kala zeera germplasm lines. The identified genotypes with high phenolic content and antioxidant activity can further be utilized in Kala zeera breeding programmes.

Mapping of quantitative trait loci for scab resistance in apple (Malus × domestica) variety, Shireen.

BACKGROUND: Scab caused by Venturia inaequalis (Cke.) Wint. is the most important fungal disease of apple. Fungicide application is a widely practiced method of disease control. However, the use of chemicals is costintensive, tedious, and ecologically unsafe. The development of genetic resistance and the breeding of resistant cultivars is the most reliable and safest option. One such source of scab resistance happens to be the variety 'Shireen', released from SKUAST-Kashmir. However, to date, the nature of resistance and its genetic control have not been characterized. Objective This research aimed to elucidate the genetic basis of scab resistance in Shireen. METHODS: Genetic mapping of quantitative trait loci (QTL) for resistance to apple scab disease was performed using an F1 cross developed between the susceptible cultivar 'StarKrimson' and the resistant cultivar 'Shireen'. The population was evaluated for two consecutive years. Further, six candidate genes were analyzed via quantitative real-time PCR, to determine their expression level in response to the pathogen infestation. RESULTS: Genotyping and disease phenotyping of populations led us to identify two quantitative trait loci (QTLs), namely qRVI.SS-LG2.2019 and qRVI.SS-LG8.2019 on chromosomes 2 and 8 with LOD-values of 7.67 and 4.99 respectively, and six potential CDGs for the polygenic resistance in 'Shireen'. The genomic region corresponding to the mapped QTLs in LG 2 and LG 8 of 'Shireen' was examined for candidate genes possibly related to scab resistance using in silico analysis. CONCLUSION: The QTLs mapped in the genetic background of Shireen are the novel QTLs and may be transferred to desirable genetic backgrounds and provide opportunities for isolation and cloning of genes apart from their utility to achieve durable resistance to scab.

Fe and Zn stress induced gene expression analysis unraveled mechanisms of mineral homeostasis in common bean (Phaseolus vulgaris L.).

Iron (Fe) and zinc (Zn) stress significantly affects fundamental metabolic and physiological processes in plants that results in reduction of plant growth and development. In the present study, common bean variety; Shalimar French Bean-1 (SFB-1) was used as an experimental material. Four different MGRL media i.e. normal MGRL medium (Control), media without Fe (0-Fe), media without Zn (0-Zn) and media with excess Zn (300-Zn) were used for growing seeds of SFB-1 under in vitro condition for three weeks under optimum conditions. Three week old shoot and root tissues were harvested from the plants grown in these four different in vitro conditions and were, subjected to Fe and Zn estimation. Further, extraction of total RNA for differential gene expression of ten candidate genes selected based on our in silico investigation and their classification, phylogeny and expression pattern was unraveled. Expression analysis of three candidate genes (OPT3, NRAMP2 and NRAMP3) in roots revealed possible cross talk among Fe/Zn stress that was further confirmed by observing less accumulation of Fe in roots under both these conditions. However, we observed, higher accumulation of Fe in shoots under 0-Fe condition compared to control that suggests precise sensing for priority based compartmentalization and partitioning leading to higher accumulation of Fe in shoots. Furthermore, the expression analysis of IRT1, FRO1 and Ferritin 1 genes under Fe/Zn stress suggested their role in uptake/transport and signaling of Fe and Zn, whereas the expression of ZIP2, NRAMP1, HA2 and GLP1 genes were highly responsive to Zn in Phaseolus vulgaris. The identified genes highly responsive to Fe and Zn stress condition can be potential candidates for overcoming mineral stress in dicot crop plants.

The S100A4 Oncoprotein Promotes Prostate Tumorigenesis in a Transgenic Mouse Model: Regulating NFκB through the RAGE Receptor.

S100A4, a calcium-binding protein, is known for its role in the metastatic spread of tumor cells, a late event of cancer disease. This is the first report showing that S100A4 is not merely a metastatic protein but also an oncoprotein that plays a critical role in the development of tumors. We earlier showed that S100A4 expression progressively increases in prostatic tissues with the advancement of prostate cancer (CaP) in TRAMP, an autochthonous mouse model. To study the functional significance of S100A4 in CaP, we generated a heterozygously deleted S100A4 (TRAMP/S100A4(+/-)) genotype by crossing TRAMP with S100A4(-/-) mice. TRAMP/S100A4(+/-) did not show a lethal phenotype, and transgenes were functional. As compared to age-matched TRAMP littermates, TRAMP/S100A4(+/-) mice exhibited 1) an increased tumor latency period (P < 0.001), 2) a 0% incidence of metastasis, and 3) reduced prostatic weights (P < 0.001). We generated S100A4-positive clones from S100A4-negative CaP cells and tested their potential. S100A4-positive tumors grew at a faster rate than S100A4-negative tumors in vitro and in a xenograft mouse model. The S100A4 protein exhibited growth factor-like properties in multimode (intracellular and extracellular) forms. We observed that 1) the growth-promoting effect of S100A4 is due to its activation of NFκB, 2) S100A4-deficient tumors exhibit reduced NFκB activity, 3) S100A4 regulates NFκB through the RAGE receptor, and 4) S100A4 and RAGE co-localize in prostatic tissues of mice. Keeping in view its growth-promoting role, we suggest that S100A4 qualifies as an excellent candidate to be exploited for therapeutic agents to treat CaP in humans.

Association between serotonin transporter gene promoter-region polymorphism and 4- and 12-week treatment response to sertraline in posttraumatic stress disorder.

BACKGROUND: We examined the association between serotonin transporter (5HTTLPR) genotype (SS vs SL vs LL) and sertraline treatment outcome in posttraumatic stress disorder (PTSD). METHODS: Outpatients (n=330) with PTSD underwent 5HTTLPR genotyping. All patients received sertraline (100 mg/day) for 12 weeks. Patients were assessed using the Clinician-Administered PTSD Scale (CAPS) and other instruments. Patients and rater were blind to the genotyping results. The primary outcome was completer sample CAPS improvement at 12 weeks. Response was defined as ≥30% improvement in CAPS total score with a CGI-I score of 1 or 2. RESULTS: The discontinuation rate was 31.5%. Adverse events led to drop out in 18.1%, 15.3%, and 5.9% of SS, SL, and LL patients, respectively (P=0.038). Among completers, there were 95, 43, and 88 patients with the SS, SL, and LL genotypes, respectively. At endpoint, CAPS total scores improved by 26% vs 46%, respectively, in SS and SL vs LL patients (P<0.001); much of this improvement (15% vs 31% in SS and SL vs LL patients, respectively; P<0.001) was apparent by week 4. The findings were largely similar for the other outcome measures. The response rate was 0%, 0%, and 47.7% in the SS, SL, and LL groups, respectively (P<0.001). LIMITATIONS: We administered a fixed dose of sertraline. For sociopolitical reasons, we planned a completer analysis only. CONCLUSIONS: Relative to the SS and SL 5HTTLPR genotypes, the LL genotype is associated with greater responsiveness of PTSD to sertraline (100mg/day) and with lower drop out due to adverse events. The S allele is associated with a striking specificity for treatment nonresponse, as defined in this study.

TP53 - molecular soldier's mutations in bladder cancer in the Kashmiri population.

PURPOSE: We made a preliminary attempt to study mutations in exons 5-8 (the DNA binding domain) of the tumor suppressor gene TP53, in urinary bladder cancer patients from Kashmir. Further the relation of clinicopathological characteristics with mutation status was asessed. MATERIALS AND METHODS: The study population consisted of 60 patients diagnosed with transitional cell carcinomas who underwent transurethral resection and /or radical cystectomy. Mutations in 5-8 exons of TP53 gene were detected by means of single strand conformation polymorphism (SSCP). All samples which showed different migration bands in SSCP were confirmed by DNA sequencing. RESULTS: 19 of 60 (31.6%) bladder cancers had mutations of the TP53 gene (11 transitions and 8 transversions), three were G→A transitions, two G→T transversions, three A→C transversions, five C→T transitions and six A→T transversions. Predominantly missense mutations (66%) were detected but no deletions or insertions were found. Statistically significant associations (< 0.05) were noted with higher tumor stage (T2 or higher), recurrence and large tumor size (> 3 cm). No correlation was found between smoking and tumor grade and the presence of TP53 mutations. CONCLUSIONS: Mutation of the TP53 gene is one of the commonest genetic changes in human bladder cancer, also in a high risk ethnic Kashmiri population.

Genetic aberrations of the K-ras proto-oncogene in bladder cancer in Kashmiri population.

PURPOSE: To assess the frequency of specific point mutations in the K-ras gene in a group of Kashmiri patients with bladder cancer. MATERIALS AND METHODS: We analyzed the incidence of K-ras exon 1 gene mutations in tumors and surgical margins in 60 patients with transitional cell carcinoma of varied clinical stages and histological grades using the polymerase chain reaction-single strand conformation polymorphism and DNA sequencing. RESULTS: A significant correlation was found between the K-ras, the lymph node status, and tumor recurrence (P < 0.05). Also, smokers and patients with higher tumor grade showed a significantly higher relative risk of developing K-ras mutations than the normal ones. CONCLUSION: K-ras exon 1 gene mutations were found with low frequency in the bladder cancer tumors from Kashmir valley, which suggests that K-ras gene might be involved in a sub-set of bladder tumors, but it needs further investigation on a larger cohort sample to authenticate the current findings.

Vehicles and ways for efficient nuclear transformation in plants.

Transgenic science and technology are fundamental to the state-of-art plant molecular genetics and crop improvement. The new generation of technology endeavors to introduce genes 'stably' into 'site-specific' locations and in 'single copy' without the integration of extraneous vector 'backbone' sequences or 'selectable markers'. Numerous plant transformation technologies have developed with the aim of achieving these objectives. Here we discuss some of these technologies, which can push the development of 'better transgenic plants with desirable characters only'.

Fisetin, a natural flavonoid, targets chemoresistant human pancreatic cancer AsPC-1 cells through DR3-mediated inhibition of NF-kappaB.

Death receptors of the tumor necrosis factor (TNF) receptor super family have been implicated in constitutive activation of nuclear factor-kappa B (NF-kappaB) in pancreatic cancer (PaC) cells. In this study, we demonstrate that fisetin, a natural flavonoid, induces apoptosis and inhibits invasion of chemoresistant PaC AsPC-1 cells through suppression of DR3-mediated NF-kappaB activation. Fisetin treatment resulted in dose-dependent inhibition of PaC cell growth and cell proliferation with concomitant induction of apoptosis. A cDNA array analysis revealed that fisetin modulates expression of more than 20 genes at transcription level with maximum decrease observed in DR3 expression and a parallel increase observed in the expression levels of IkappaBalpha, an NF-kappaB inhibitor. Down-regulation of DR3 in PaC cells was found to down regulate activated pNF-kappaB/p65, pIkBalpha/beta kinases (pIKK's), MMP9 and XIAP that mostly impart chemoresistance in PaC. Immunoblotting and EMSA analysis showed a marked decrease in pNF-kappaB and NF-kappaB DNA binding activity, respectively, with modest decrease in NF-kappaB promoter activity and significant decrease in MMP9 promoter activity with fisetin treatment. Importantly, consistent with these findings, we further found that transient down-regulation of DR3 by RNA interference significantly augmented fisetin induced changes in cell proliferation, cell invasion and apoptosis paralleled with decrease in pNF-kappaB, pIKKalpha/beta, MMP9, XIAP and NF-kappaB DNA binding activity. Blocking of DR3 receptor with an extra cellular domain blocking antibody demonstrated similar effects. These data provide evidence that fisetin could provide a biological rationale for treatment of pancreatic cancer or as an adjuvant with conventional therapeutic regimens.

Lupeol triterpene, a novel diet-based microtubule targeting agent: disrupts survivin/cFLIP activation in prostate cancer cells.

Recently we showed Lupeol, a triterpene, found in fruits and vegetables inhibits the growth of tumors originated from human androgen-sensitive prostate cancer (CaP) cells and decreases the serum-PSA levels in a mouse model. Here, we provide evidence that Lupeol inhibits the growth of androgen-sensitive as well as androgen-insensitive CaP cells by inducing G2/M cell cycle arrest without exhibiting any toxicity to normal human prostate epithelial cells (PrEC) at the doses at which it kills cancer cells. We observed that Lupeol treatment to LNCaP and DU145 cells resulted in a dose-dependent (i) decrease in the protein levels of Cyclins-A, -B1, -D1, -D2, -E2, cyclin-dependent kinase (cdk)-2 and (ii) increase in the protein level of CDK-inhibitor p21. Since G2/M cell cycle phase is regulated by microtubule assembly, we investigated effect of Lupeol on microtubule assembly, its regulation and down-stream targets in CaP cells. Lupeol treatment significantly modulated the level of (i) microtubule components alpha-tubulin and beta-tubulin, (ii) microtubule-regulatory protein stathmin, and (iii) microtubule-regulatory down-stream target/pro-survival protein survivin. Lupeol treatment also decreased the level of anti-apoptotic protein cFLIP. Finally, Lupeol was observed to significantly decrease the transcriptional activation of survivin and cFLIP genes in CaP cells. We conclude that the Lupeol-induced growth inhibition of CaP cells is a net outcome of simultaneous effects on stathmin, cFLIP, and survivin which results in the disruption of microtubule assembly. We suggest that Lupeol alone or as an adjuvant to other microtubule agents could be developed as a potential agent for the treatment of human CaP.

Lupeol inhibits proliferation of human prostate cancer cells by targeting beta-catenin signaling.

Lupeol, a dietary triterpene, was shown to decrease serum prostate-specific antigen levels and inhibit the tumorigenicity of prostate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibits the proliferative potential of CaP cells and delineated its mechanism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates the expression level of genes such as ERBB2, tissue inhibitor of metalloproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2 that are known to be associated with proliferation and survival. A common feature of these genes is that all of them are known to either regulate or act as downstream target of beta-catenin signaling that is highly aberrant in CaP patients. Lupeol treatment significantly (1) reduced levels of beta-catenin in the cytoplasmic and nuclear fractions, (2) modulated expression levels of glycogen synthase kinase 3 beta (GSK3beta)-axin complex (regulator of beta-catenin stability), (3) decreased the expression level and enzymatic activity of MMP-2 (downstream target of beta-catenin), (4) reduced the transcriptional activation of T Cell Factor (TCF) responsive element (marker for beta-catenin signaling) in pTK-TCF-Luc-transfected cells and (5) decreased the transcriptional activation of MMP-2 gene in pGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatment on beta-catenin degradation were significantly reduced in CaP cells where axin is knocked down through small interfering RNA transfection and GSK3beta activity is blocked. Collectively, these data suggest the multitarget efficacy of Lupeol on beta-catenin-signaling network thus resulting in the inhibition CaP cell proliferation. We suggest that Lupeol could be developed as an agent for chemoprevention as well as chemotherapy of human CaP.

Suppression of cFLIP by lupeol, a dietary triterpene, is sufficient to overcome resistance to TRAIL-mediated apoptosis in chemoresistant human pancreatic cancer cells.

Overexpression of cellular FLICE-like inhibitory protein (cFLIP) is reported to confer chemoresistance in pancreatic cancer (PaC) cells. This study was designed to investigate the effect of lupeol, a dietary triterpene, on (a) apoptosis of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy-resistant PaC cells overexpressing cFLIP and (b) growth of human pancreatic tumor xenografts in vivo. The effect of lupeol treatment on proliferation and TRAIL/caspase-8/cFLIP machinery in PaC cells was investigated. Next, cFLIP-overexpressing and cFLIP-suppressed cells were tested for sensitivity to recombinant TRAIL therapy in the presence of lupeol. Further, athymic nude mice implanted with AsPC-1 cells were treated with lupeol (40 mg/kg) thrice a week and surrogate biomarkers were evaluated in tumors. Lupeol alone treatment of cells caused (a) decrease in proliferation, (b) induction of caspase-8 and poly(ADP-ribose) polymerase cleavage, and (c) down-regulation of transcriptional activation and expression of cFLIP. Lupeol was observed to increase the TRAIL protein level in cells. Lupeol significantly decreased the viability of AsPC-1 cells both in cFLIP-suppressed cells and in cFLIP-overexpressing cells. Lupeol significantly sensitized chemoresistant PaC cells to undergo apoptosis by recombinant TRAIL. Finally, lupeol significantly reduced the growth of human PaC tumors propagated in athymic nude mice and caused modulation of cFLIP and TRAIL protein levels in tumors. Our findings showed the anticancer efficacy of lupeol with mechanistic rationale against highly chemoresistant human PaC cells. We suggest that lupeol, alone or as an adjuvant to current therapies, could be useful for the management of human PaC.

Delphinidin, a dietary anthocyanidin in pigmented fruits and vegetables: a new weapon to blunt prostate cancer growth.

In a recent publication, we have shown that delphinidin, an anthocyanidin induces apoptosis and cell cycle arrest in highly metastatic human prostate cancer (PCa) PC3 cells. Extending these studies, we provide additional evidence that delphinidin induces apoptosis and cell cycle arrest in androgen refractory human PCa 22Rnu1 cells and that these effects are concomitant with inhibition of NFkappaB. We observed that delphinidin treatment to 22Rnu1 cells resulted in a dose-dependent (i) G(2)/M phase cell cycle arrest, (ii) induction of apoptosis (iii) and inhibition of NFkappaB signaling. The induction of apoptosis by delphinidin was mediated via activation of caspases since a general caspase inhibitor Z-VAD-FMK significantly reversed this effect. Delphinidin treatment to cells resulted in a dose-dependent decrease in (i) phosphorylation of IKKgamma (NEMO), (ii) phosphorylation of NFkappaB inhibitory protein IkappaBalpha, (iii) phosphorylation of NFkappaB/p65 at Ser(536) and NFkappaB/p50 at Ser529, (iv) NFkappaB/p65 nuclear translocation, and (v) NFkappaB DNA binding activity. Taken together, our data show that delphinidin induces apoptosis of both androgen independent and androgen refractory human PCa cells via activation of caspases and in addition, this effect might be due to inhibition of NFkappaB signaling. We suggest that delphinidin could be developed as a novel agent against PCa.

A study on p53 gene alterations in esophageal squamous cell carcinoma and their correlation to common dietary risk factors among population of the Kashmir valley.

AIM: To systematically examine the extent of correlation of risk factors, such as age, consumed dietary habit and familial predisposition with somatic Tp53 molecular lesion causal to elevate carcinogenesis severity of esophageal squamous cell carcinoma (ESCC) among the Kashmiri population of Northern India. METHODS: All cases (n = 51) and controls (n = 150) were permanent residents of the Kashmir valley. Genetic alterations were determined in exons 5-8 of Tp53 tumor suppressor gene among 45 ESCC cases histologically confirmed by PCR-SSCP analysis. Data for individual cancer cases (n = 45) and inpatient controls (n = 150) with non-cancer disease included information on family history of cancer, thirty prevailing common dietary risk factors along with patient's age group. Correlation of genetic lesion in p53 exons to animistic data from these parameters was generated by Chi-square test to all 45 histologically confirmed ESCC cases along with healthy controls. RESULTS: Thirty-five of 45 (77.8%) histologically characterized tumor samples had analogous somatic mutation as opposed to 1 of 45 normal sample obtained from adjacent region from the same patient showed germline mutation. The SSCP analysis demonstrated that most common p53 gene alterations were found in exon 6 (77.7%), that did not correlate with the age of the individual and clinicopathological parameters but showed significant concordance (P<0.05) with familial history of cancer (CD = 58), suggesting germline predisposition at an unknown locus, and dietary habit of consuming locally grown Brassica vegetable "Hakh" (CD = 19.5), red chillies (CD = 20.2), hot salty soda tea (CD = 2.37) and local baked bread (CD = 1.1). CONCLUSION: Our study suggests that somatic chromosomal mutations, especially in exon 6 of Tp53 gene, among esophageal cancer patients of an ethnically homogenous population of Kashmir valley are closely related to continued exposure to various common dietary risk factors, especially hot salty tea, meat, baked bread and "Hakh", that are rich in nitrosoamines and familial cancer history.

A preliminary investigation demonstrating the effect of quercetin on the expression of genes related to cell-cycle arrest, apoptosis and xenobiotic metabolism in human CO115 colon-adenocarcinoma cells using DNA microarray.

The role of the natural dietary flavonoid chemical quercetin (an antioxidant) in the prevention and treatment of colon cancer is receiving a great deal of attention. However, little is known about the molecular mechanisms of action of this flavonoid. In the present study, whole genome DNA microarrays were used to evaluate the effect of quercetin on gene expression in the CO115 colon-adenocarcinoma cell line with the completely deleted chromosome 18 harbouring the SMAD4 tumour-suppressor gene related to colon carcinogenesis. The study demonstrated that quercetin, widely present in fruit and vegetables, inhibited the growth of CO115 cells at 100 microM concentration in both the G(1)/S and the G(2)/M phases by modulating cell-cycle and apoptosis-related genes. Differential changes in accumulation of transcripts analysed for cells treated with 100 microM quercetin for 24 and 48 h in three independent repeated experiments revealed 5060-7000 differentially expressed genes. This means that quercetin probably does have a broad modulatory effect on gene expression in colon cancer. Out of these differentially expressed genes, the expression of 35 and 23 unique set of genes involved in cell-cycle control, apoptosis and xenobiotic metabolism were significantly altered after 24 and 48 h quercetin treatment respectively. Our results represent a novel aspect of the biological profile of quercetin that induces cell-cycle arrest through modulation of cell-cycle-related and apoptosis genes. The present study demonstrates a new step in elucidating the underlying molecular mechanisms of the antitumour action of quercetin, which could become a chemopreventive or chemotherapeutic agent for colon cancer.

Molecular cloning and genetic analysis of functional merB gene from indian isolates of Escherichia coli.

Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD3 merB and pIAAD14 merB showed slight variation (2%) at base. However, this variation in pIAAD3 due to the absence of base "T" at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD3 merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5alpha E. coli cells possessing pIAAD3 merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD3 merB for the bioremediation of mercury-polluted sites.