Hexane Extract of Garcinia quaesita Fruits Induces Apoptosis in Breast Cancer Stem Cells Isolated from Triple Negative Breast Cancer Cell Line MDA-MB-231.

Development of therapy resistance is a major clinical issue in breast cancer treatments. Breast cancer stem cells (bCSCs) have a clearly defined role in the development of breast cancer therapy resistance and tumor recurrence. Therefore, discovery of new treatment strategies to circumvent cancer therapy resistance and tumor recurrence by targeting bCSCs is desperately needed. Fruits of many Garcinia species are edible and, possess a range of health benefits. Garcinia quaesita, a species in the genus Garcinia, is endemic to Sri Lanka. Dried fruits of G. quaesita are commonly used to flavor dishes in Sri Lanka. The present study assessed the potential anticancer and apoptotic properties of G. quaesita fruit extracts in bCSCs using WST-1 cell proliferation assay, sphere formation assay, caspase 3/7 assay, real-time PCR and fluorescent and phase-contrast microscopy. DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate), ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) and FRAP (Ferric Reducing Anti-oxidant Power) assays were used as anti-oxidant assays. The hexane extract of G. quaesita fruits was found to mediate cytotoxicity in bCSCs through induction of apoptosis. Furthermore, the hexane extract showed free radical scavenging ability. This pilot investigation provides a rationale to consume G. quaesita fruits as an anticancer dietary supplement for breast cancer patients.

Role of the PI3K/AKT/mTOR signaling pathway in ovarian cancer: Biological and therapeutic significance.

Ovarian cancer (OC) is a lethal gynecological cancer. The phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway plays an important role in the regulation of cell survival, growth, and proliferation. Irregularities in the major components of the PI3K/AKT/mTOR signaling pathway are common in human cancers. Despite the availability of strong pre-clinical and clinical data of PI3K/AKT/mTOR pathway inhibitors in OC, there is no FDA approved inhibitor available for the treatment of OC. Here, we outline the importance of PI3K/AKT/mTOR signaling pathway in OC tumorigenesis, proliferation and progression, and pre-clinical and clinical experience with several PI3K/AKT/mTOR pathway inhibitors in OC.

Pattern of nucleotide variants of TP53 and their correlation with the expression of p53 and its downstream proteins in a Sri Lankan cohort of breast and colorectal cancer patients.

BACKGROUND: Breast cancer (BC) is known to be the most common malignancy in females whereas colorectal cancer (CRC) incidence also higher in both genders in Sri Lanka. TP53 is an important tumour suppressor gene and its somatic mutations are reported in approximately 27% of BC and 43% of CRC cases. Analysis of TP53 gene variants not only provides clues for the aetiology of the tumour formation, but also has an impact on treatment efficacy. The current study was conducted to investigate the pattern of TP53 variants in patients with BC and CRC from Sri Lanka. METHODS: 30 patients with BC, 21 patients with CRC and an equal number of healthy controls were screened for mutational status of TP53 by polymerase chain reaction (PCR) followed by direct sequencing. In addition, a subset of these samples were analysed for the protein expression of p53 and comparison made with the mutational status of TP53. We also analysed the protein expression of p21 and MDM2 as potential indicators of p53 functional status and compared it with the protein expression of p53. Additionally, hotspot codons of the KRAS, BRAF and PIK3CA genes were also analysed in a subset of CRC patients. RESULTS: Twenty seven sequence variants, including several novel variants in the TP53 gene were found. Nine BC and seven CRC tumour samples carried pathogenic TP53 variants. Pathogenic point missense variants were associated with strong and diffuse positive staining for p53 by immunohistochemistry (IHC), whereas, wild type TP53 showed complete absence of positive IHC staining or rare positive cells, regardless of the type of cancer. There was no direct correlation between p21 or MDM2 expression and p53 expression in either BCs or CRCs. Four of the CRC patients had pathogenic hotspot variants in KRAS; three of them were on codon 12 and one was on codon 61. CONCLUSION: The prevalence of pathogenic somatic TP53 variants was 31 and 33.33% in the studied BC and CRC cohorts respectively. All of them were located in exons 5-8 and the pathogenic missense variants were associated with strong immuno-positive staining for p53.

In vitro assays and techniques utilized in anticancer drug discovery.

Development of a cancer is a multistep process and six major hallmarks of cancer that are known to control malignant transformation have been described. Anticancer drug development is a tedious process, requiring a number of in vitro, in vivo and clinical studies. In vitro assays provide an initial platform for cancer drug discovery approaches. A wide range of in vitro assays/techniques have been developed to evaluate each hallmark feature of cancer and selection of a particular in vitro assay or technique mainly depends on the specific research question (s) to be examined. In the present review, we have described some commonly utilized in vitro assays and techniques used to examine cell viability/proliferation, apoptosis, cellular senescence, invasion and migration, oxidative stress and antioxidant effects, gene and protein expression, angiogenesis and genomic alterations in cancer drug discovery. Additionally, uses of modern techniques such as high throughput screening, high content screening and reporter gene assays in cancer drug discovery have also been described.

Circulating leptin, soluble leptin receptor, free leptin index, visfatin and selected leptin and leptin receptor gene polymorphisms in sporadic breast cancer.

Leptin and visfatin are implicated in breast cancer risk but studies accounting for bioavailability of leptin are sparse. Reports on the association of leptin gene (LEP) and leptin receptor gene (LEPR) polymorphisms with breast cancer are also inconsistent. Only a very few studies have examined biochemical and genetic variables concomitantly in the same cohort. A matched pairs study was carried out to ascertain whether plasma leptin, soluble leptin receptor, free leptin index (leptin/soluble leptin receptor), serum visfatin and selected LEP and LEPR polymorphisms are risk factors for sporadic breast cancer. Newly diagnosed sporadic breast cancer patients (N=80) were matched for age, body mass index (BMI) and menopausal status with healthy controls. Plasma leptin, soluble leptin receptor and serum visfatin were measured by enzyme-immunoassay. LEP -2548 A/G and LEPR K109R, LEPR Q223R polymorphisms were determined by genotyping. Leptin (p=0.0234), leptin/BMI (p=0.0468), free leptin index (p<0.0001) and visfatin (p=0.0002) were significantly higher and soluble leptin receptor (p<0.0001) was significantly lower in patients. LEPR gene K109R A/G polymorphism increased breast cancer risk (odds ratio: 4.125). Multivariate analysis confirmed that leptin, soluble leptin receptor, free leptin index and G109 (R109) allele of the LEPR gene K109R polymorphism are risk factors for breast cancer. When stratified by menopausal status free leptin index and soluble leptin receptor remained as risk factors irrespective of menopausal status while LEPR gene K109R A/G polymorphism remained as a risk factor only in the postmenopausal group.

In silico identification and in vitro validation of alpha-hederin as a potent inhibitor of Wnt/ß-catenin signaling pathway in breast cancer stem cells.

Cancer stem cells (CSCs) play a vital role in metastasis, recurrence and chemoresistance in breast cancer. ß-catenin, which is a frequently over activated protein in CSCs, binds to T-cell factor/lymphoid enhancer factor (Tcf/Lef) family transcription factors leading to ectopic expression of Wnt pathway responsive genes necessary for the maintenance and action of CSCs. With the aim of identifying a small molecules that can effectively eliminate CSCs, molecular docking studies were performed against the Tcf/Lef binding hotspot on ß-catenin using a library of 100 natural or synthetic small molecules. Small molecule ligands giving docking energy better than - 7 kcal/mol were further investigated by binding interactions analysis and molecular dynamics (MD) simulations. These compounds were then investigated in vitro, for cytotoxicity against CSCs isolated from MDA-MB-231 triple negative breast cancer cells. Alpha-hederin (AH) was identified as the only compound in the selected library that has cytotoxicity against breast CSCs. AH was further investigated for it's ability to regulate Wnt pathway target genes (Cyclin D1 and CD44)and the tumor suppressor p53by real-time quantitative PCR. Absorption, distribution, metabolism, excretion and toxicity properties of the AH was predicted in silico. AH significantly down regulated the transcription of Cyclin D1 and CD44 while up-regulating the transcription of p53. AH was predicted to have acceptable drug likeness. Although AH is currently known to inhibit the growth of various cancer cells in vitro, present study demonstrated for the first time that it is a potent inhibitor of Wnt/ß-catenin signaling pathway and induce apoptosis in breast CSCs.

Novel gross deletion at the LHX4 gene locus in a child with growth hormone deficiency.

OBJECTIVE: To identify and characterize a novel deletion at the LHX4 gene locus in a proband with growth hormone deficiency (GHD). METHODS: Long range polymerase chain reaction (PCR) amplification was used to confirm the suspected deletion and to identify the rough locations of the end points. Sanger sequencing was carried out to identify the exact end points of the deletion. RESULTS: Suspected deletion was confirmed via long range PCR amplification. Sanger sequencing identified the end points of the deletion within three nucleotide repeat sequences ("CTT"). The total length of the deleted segment was 12 127 base pairs and it includes complete exon 5 and exon 6 of the LHX4 gene. Therefore the homeodomain motif coded by exons 4 and 5, might be affected. CONCLUSION: We have identified a novel deletion that spans exon 5 and exon 6 of the LHX4 gene that could have occurred via microhomology mediated non-recurrent rearrangement. The deletion characterized does not appear to have been reported before. To our knowledge this novel deletion is the first identified LHX4 variant from Sri Lanka and it explains the phenotype of the proband characterized by growth hormone deficiency, hypoplastic anterior pituitary and subsequent deficiency of thyroid stimulating hormone and adrenocorticotropic hormone (ACTH).

Cytotoxicity against Human Hepatocellular Carcinoma (HepG2) Cells and Anti-Oxidant Activity of Selected Endemic or Medicinal Plants in Sri Lanka.

Hepatocellular carcinoma (HCC) is the most fatal cancer globally with limited treatment options. Plants and herbs have been used to treat cancer and other diseases for a long time by traditional practitioners in Sri Lanka. In the present study, leaf and bark extracts of selected plants were investigated for cytotoxic properties on HepG2 cells. Anti-oxidant activity and total phenolic and flavonoid contents were also determined. Plant extracts that exerted cytotoxic effects on the HepG2 cell line with IC50 <100 µg/mL were tested on normal liver epithelial cells (THLE-3). Out of the 56 extracts, 21 exhibited potent cytotoxic effects (IC50 < 100 µg/mL) on HepG2 cells after 48 h exposure, and 12 were less toxic (IC50 > 100 µg/mL) to THLE-3 normal liver cells. Six extracts exhibited potent radical scavenging activity with EC50 < 100 µg/mL against 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, while 17 extracts showed potent anti-oxidant activity (Trolox equivalents > 100 mg/g) against ferric reducing anti-oxidant power (FRAP) assay. Out of the 56 extracts, 15 had total phenolic content above 100 mg/g of gallic acid equivalents, and 4 had flavonoid content above 100 mg/g of quercetin equivalents. Among the extracts screened, hexane, dichloromethane, ethyl acetate, and methanol extracts of Allophylus cobbe leaves (IC50 - 9.388, 6.8, 19.95, and 11.3 µg/mL, respectively), Madhuca longiflora bark (IC50 - 14.42 µg/mL), methanol extract of Munronia pinnata bark (IC50 - 52.06 µg/mL), and hexane, dichloromethane, ethyl acetate, and methanol extracts of Adenanthera bicolor (IC50 - 45.86, 27.35, 24.56, and 61.83 µg/mL, respectively) exerted potent cytotoxicity against HepG2 with less toxicity (IC50 > 100 µg/mL) to THLE-3 cells after 48 h of incubation. These findings provide a direction to isolate possible anti-cancer compounds for hepatocellular carcinoma.

Identification of 3-O-α-l-arabinosyl oleanolic acid, a triterpenoid saponin, as a new breast cancer stem cell growth inhibitor.

This study was conducted to determine the anti-cancer activity of 3-O-α-L-arabinosyl oleanolic acid (3-O-L-AO), a triterpenoid saponin, isolated from the leaves of Schumacheria castaneifolia Vahl in breast cancer stem cells (bCSCs) grown in hypoxia. Anti-proliferative effects of 3-O-L-AO in bCSCs were determined using WST-1 assay. Real-time PCR was employed to evaluate the effects of 3-O-L-AO on apoptosis. Compound 3-O-L-AO exerted greater anti-proliferative effect in bCSCs grown under hypoxic conditions. Treatment of bCSCs with 3-O-L-AO resulted in a significant up-regulation of Bax and p53 and a significant down-regulation of survivin, HIF-1α and HIF-2α. Activation of caspase 3/7 activity and apoptosis-related morphological changes in bCSCs exposed to 3-O-L-AO further confirmed that 3-O-L-AO can induce apoptosis. Collectively, the results obtained indicated that 3-O-L-AO can be considered as a new anti-cancer agent to target chemo- and radio-therapy-resistant bCSCs.

A new liposomal nanocarrier for co-delivery of gedunin and p-glycoprotein siRNA to target breast cancer stem cells.

Gedunin is a secondary metabolite found in neem tree. Since the first discovery of this compound, its bio-active properties have been continuously evaluated. However, the low hydrophobicity of gedunin decreases its bioavailability and pharmacokinetic profile. In the present investigation, a new liposomal nanocarrier for co-delivery of gedunin and P-glycoprotein (P-gp) siRNA [siRNA coated liposomal gedunin (Lipo-Ged-siRNA)] was developed to improve the anti-proliferative activity of gedunin. Characteristics of prepared Lipo-Ged-siRNA demonstrated promising effects. Lipo-Ged-siRNA showed greater anti-proliferative effects (IC50-8.5 µg/mL) followed by pure gedunin (IC50- 40.2 µg/mL) in breast cancer stem cells (bCSCs). Immunofluorescence analysis demonstrated reduced expression of P-gp following exposure to Lipo-Ged-siRNA. Furthermore, Lipo-Ged-siRNA affected the expression of ABCB1, Cyclin D1, Bax, p53, and surviving genes in bCSCs.

Interaction of Gut Microbiome and Host microRNAs with the Occurrence of Colorectal and Breast Cancer and Their Impact on Patient Immunity.

Breast and colorectal cancers are two primary malignancies on which most of the research done worldwide investigates the potential genetic and environmental risk factors and thereby tries to develop therapeutic methods to improve prognosis. Breast cancer is the most diagnosed cancer type in women, while colorectal cancer is diagnosed in males as the third most and females as the second most cancer type. Though these two cancer types are predominantly seen in adult patients worldwide, in the current context, these malignancies are diagnosed at a younger age with a significant rate of incidents than previous. Such early-onset cancers are generally present at an advanced stage of the most aggressive type with a poor prognosis. In the past, the focus of the research was mainly on studying possible candidate genes to understand the onset. However, it is now recognized that genetics, epigenetics, and other environmental factors play a pivotal role in cancer susceptibility. Thus, most studies were diversified to study the behavior of host microRNAs, and the involvement of gut microbiota and good communication between them surfaced in the occurrence and state of the disease. It is understood that the impact of these factors affects the outcome of the disease. Out of the adverse outcomes identified relating to the disease, immunosuppression is one of the most concerning outcomes in the current world, where such individuals remain vulnerable to infections. Recent studies revealed that microbiome and microRNA could create a considerable impact on immunosuppression. This review focused on the behavior of host microRNAs and gut microbiome for the onset of the disease and progression, thereby influencing an individual's immunosuppression. Understanding the interactions among microRNA, microbiome, presentation of the disease, and impact on the immune system will be immensely useful for developing future therapeutic strategies based on targeting host microRNA and the patient's gut microbiome. Therapies such as inhibitory-miRNA therapies, miRNA mimic-based therapeutics, immune checkpoint blockade therapies, and bacteria-assisted tumor-targeted therapies help modulate cancer. At the same time, it paid equal attention to potential noninvasive biomarkers in diagnosis, prognosis, and therapeutics in both cancers.

Evaluation of non-coding region sequence variants and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in individuals of Sri Lankan Sinhalese ethnicity.

Mitochondrial DNA (mtDNA) mutations have been reported to be associated with various diseases, including cancer. The present study investigated the mtDNA non-coding region mutations and mitochondrial haplogroups as potential biomarkers of sporadic breast cancer in Sri Lankan Sinhalese women. Mitochondrial macro-haplogroups were determined using PCR-restriction fragment length polymorphism, whereas non-coding region sequences were determined using Sanger sequencing. The sequence of the non-coding region was also used to confirm haplogroup status. Neither the mutations in the non-coding region nor the mitochondrial haplogroups that were reported as risk factors in other populations, were determined to be potential risk factors for sporadic breast cancer in the present study. Furthermore, several novel mutations were identified in the present matched pairs case-controlled study. The M65a haplogroup with an additional mutation at position 16311 (P=0.0771) and mutations at the ori-b site (P=0.05) were considered a weak risk factor and protective factor, respectively, for sporadic breast cancer in Sinhalese women. Previous studies have indicated the use of mtDNA mutations as a biomarker; however, the present study showed that such biomarkers need to be validated for individual ethnic groups before they can be recommended for use in the prediction of disease.

Overview of the genetic basis toward early detection of breast cancer.

Cancer is a socioeconomical burden in any nation. Out of that, breast cancer is identified as the most common malignancy worldwide among women irrespective of age. As women are an important segment in a community, the weakening of their strength toward the development of a nation is a critical problem in each nation. In this review, it was aimed to discuss the characteristics of cancer genome, cancer genetics, and cancer epigenetics in general and then focus on discussing both genetic and nongenetic factors responsible for the predisposition of breast cancer in humans. More emphasis was placed on genes responsible for the early onset of the disease and which can be used as genetic tools in the identification of the disease at an early stage. Then the context of genetic involvement toward the breast cancer occurrence before age of 40 years was highlighted accordingly. In addition to genetic testing, the review paid adequate attention to mention novel liquid biopsy techniques and other clinical, laboratory, and radiologic assessments. These techniques can be used in early detection and recurrence as well as the surveillance of the patients after primary therapies.

Emerging role of histone deacetylase inhibitors as anti-breast-cancer agents.

Breast cancer (BC) remains the most frequently diagnosed cancer in women. A balance in the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is necessary for epigenetic regulation of gene expression. Impairment in the balance between the actions of HATs and HDACs has been reported in the development of BC. By targeting histone and several non-histone proteins, histone deacetylase inhibitors (HDACi) can maintain the cellular acetylation profile and reverse the function of several proteins responsible for BC development. Preclinical and clinical data show that HDACi can evoke different anticancer mechanisms in distinct BC types.

Nucleotide variants and protein expression of TP53 in a Sri Lankan cohort of patients with head and neck cancer.

Head and neck cancer (HNC) is the leading cancer in Sri Lankan males and second most common cancer among Sri Lankan females. This is the first study, to the best of our knowledge, that has focused on investigating the association between TP53 somatic DNA variants, with p53 protein expression and risk factors in a cohort of Sri Lankan patients with HNC. A total of 44 patients with cancer and 20 healthy controls were studied. In total, 36 genomic DNA sequence variants were found, including several novel variants (two deletions in exons 4 and 6, two in the 3' untranslated region and several intronic variants). A total of 14 tumour samples carried pathogenic TP53 mutations. A random selection of 24 samples was analysed immunohistochemically for p53 protein expression. All the samples with point missense variants were strongly immuno­positive, whereas, samples with nonsense and frameshift TP53 variants were immuno­negative for p53 immunohistochemical staining. Although, the human papilloma virus is a known risk factor for HNC, results from the present study identified an absence or lower level of infection in the Sri Lankan cohort.

Novel gross deletion at the GHRHR gene locus possibly mediated by Alu specific microhomology identified in a Sri Lankan patient with isolated growth hormone deficiency.

OBJECTIVE: Characterization of a deletion in the exon 1 and 5' regulatory region of the GHRHR gene in a proband with isolated growth hormone deficiency. METHODS: Multiple ligation dependent probe amplification (MLPA) assay was carried out to confirm the homozygous deletion which was suspected during screening of the GHRHR gene by single strand conformation polymorphism. A series of short range PCR amplifications were carried out to map the approximate location of the break points of the deletion. Sanger sequencing was carried out to locate the break points and to identify the length of the deletion. Long range PCR amplification was carried out to confirm the length of the deletion and to screen the parents of the proband for the deletion. RESULTS: A homozygous deletion was confirmed via MLPA assay. Zones of sequence similarity between upstream intergenic region and intron 1 of the GHRHR gene were identified. Break points of the deletion were identified within perfectly matching 32 bp repeat sequences ie: microhomologies in the specified zones. The novel deletion may have arisen via Alu specific microhomology mediated non-recurrent rearrangement in the maternal lineage of the proband. The deletion being reported in this study include, last 3118 bp from the upstream intergenic region and complete exon 1 and first 2620 bp from intron 1 and one of the 32 bp microhomologies. The total length of the deleted segment was 5875 bp. As the deleted region contained significant elements essential for gene expression, the identified deletion is being reported as likely pathogenic. The same deletion was identified in the mother in heterozygous state. CONCLUSION: We have characterized a novel deletion that seems to have arisen via Alu specific microhomology mediated non-recurrent rearrangement at GHRHR gene locus. HGVS nomenclature of the deletion is c.-3166_58-2057del. This novel structural variant was identified to be the cause of IGHD of the affected proband.

A Review on Ethnopharmacological Applications, Pharmacological Activities, and Bioactive Compounds of Mangifera indica (Mango).

Mangifera indica (family Anacardiaceae), commonly known as mango, is a pharmacologically, ethnomedically, and phytochemically diverse plant. Various parts of M. indica tree have been used in traditional medicine for the treatment of different ailments, and a number of bioactive phytochemical constituents of M. indica have been reported, namely, polyphenols, terpenes, sterols, carotenoids, vitamins, and amino acids, and so forth. Several studies have proven the pharmacological potential of different parts of mango trees such as leaves, bark, fruit peel and flesh, roots, and flowers as anticancer, anti-inflammatory, antidiabetic, antioxidant, antibacterial, antifungal, anthelmintic, gastroprotective, hepatoprotective, immunomodulatory, antiplasmodial, and antihyperlipemic. In the present review, a comprehensive study on ethnopharmacological applications, pharmacological activities, and bioactive compounds of M. indica has been described.

Protective Effects of Six Selected Dietary Compounds against Leptin-Induced Proliferation of Oestrogen Receptor Positive (MCF-7) Breast Cancer Cells.

BACKGROUND: Obesity is considered as one of the risk factors for breast cancer. Leptin has been found to be involved in breast cancer progression. Therefore, novel approaches to antagonize biological effects of leptin are much needed. The objective of this study was to evaluate the protective effects of six dietary compounds (quercetin, curcumin, gallic acid, epigallocatechin gallate (EGCG), ascorbic acid and catechin) and assess the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in leptin-stimulated MCF-7 breast cancer cells in vitro. METHODS: MCF-7 cells were exposed to leptin, leptin and compound and compound alone for 48 h. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT and fluorometric assays after 48 h incubation. Phosphorylation of ERK1/2 was quantified by ELISA. RESULTS: Only quercetin, curcumin and EGCG showed significant protective effects against leptin-induced proliferation of MCF-7 cells. Increase in ERK1/2 phosphorylation in response to leptin was reduced by the addition of quercetin, curcumin and EGCG. CONCLUSIONS: Considering the high prevalence of obesity, this observation provides a rationale for use of curcumin, quercetin and EGCG as antagonists of leptin in the treatment of obese breast cancer patients.

Isolation of a new resorcinolic lipid from Mangifera zeylanica Hook.f. bark and its cytotoxic and apoptotic potential.

Mangifera zeylanica is a plant endemic to Sri Lanka and its bark has been used in traditional medicine to treat some cancers. This study was aimed to isolate potentially cytotoxic compound/s from the hexane extract of the bark of M. zeylanica by bio-activity guided fractionation. The structure of the isolated compound (1) was elucidated using 1H, 13C NMR and mass spectrometric techniques. Compound 1 was identified as a new resorcinolic lipid (5-((8Z, 11Z, 14Z)-hexatriaconta-8, 11, 14-trienyl) benzene-1,3-diol). Apoptotic potential of the isolated compound was determined only in MCF-7 (estrogen receptor positive) breast cancer cells to which it was more cytotoxic than to normal mammary epithelial cells. Oxidative stress markers [reactive oxygen species (ROS), glutathione levels (GSH) and glutathione-S-transferase (GSH)] were also determined in MCF-7 cells treated with compound 1. Treatment with compound 1 led to an increase in caspase 7 activity, morphological features of apoptosis and DNA fragmentation in MCF-7 cells. Furthermore, it also led to an increase in ROS and GST levels while depleting GSH levels. Results of this study suggest that isolated new resorcinolic lipid can induce apoptosis in MCF-7 cells, possibly via oxidative stress mechanism.

Pathogenic and likely pathogenic genetic alterations and polymorphisms in growth hormone gene (GH1) and growth hormone releasing hormone receptor gene (GHRHR) in a cohort of isolated growth hormone deficient (IGHD) children in Sri Lanka.

OBJECTIVE: Genetic alterations in GH1 and GHRHR genes are known to cause isolated growth hormone deficiency (IGHD). Of these, GHRHR codon 72 mutation has been reported to be highly prevalent in the Indian subcontinent, but among Sri Lankans its prevalence was low compared to reports from neighboring countries. The present study was therefore carried out to identify genetic alterations in the GH1 gene and rest of the GHRHR gene in a cohort of Sri Lankan IGHD patients who tested negative for GHRHR codon 72 mutation. METHODS: Fifty five IGHD children negative for codon 72 (GHRHR) mutation were screened for gross GH1 gene deletion by polymerase chain reaction (PCR) and restriction fragment length polymorphism technique. The coding, intronic and promoter regions of the GH1 gene were sequenced in children who were negative for GH1 deletion (N=53). In a subset (N=40), coding, flanking intronic and promoter regions of the GHRHR gene were screened by single strand conformation polymorphism/sequencing. Identified coding region and intronic variants were subjected to in silico analysis to ascertain pathogenicity. Family members available were screened for the significant variants observed in the index child. RESULTS: Gross GH1 gene deletions, 6.7kb and 7.0kb were observed in one child each. One novel and 24 reported single nucleotide variants (SNVs) were observed in the GH1 gene and its promoter. These included one reported pathogenic splice site mutation (c.172-2A>T) and one reported likely pathogenic missense mutation (c.406G>T). One large novel deletion of 5875 base pairs that included exon 1, one likely pathogenic novel SNV (c.211G>T) and 18 reported SNVs were observed in the GHRHR gene. Fourteen variants observed were of uncertain significance (8 in GH1 and 6 in GHRHR), twenty three variants were likely benign (11 in GH1 and 12 in GHRHR) and four variants were benign (4 in GH1 and none in GHRHR). CONCLUSION: In a cohort of IGHD children, six pathogenic or likely pathogenic genetic alterations of either GH1 gene or GHRHR gene were found. These affected a total of six children. Pathogenic status of four of these had been reported in the literature. Novel SNV in the GHRHR gene was predicted to be pathogenic through in silico analysis. The large novel deletion is likely to be pathogenic as it included exon 1 of GHRHR gene. Analysis of other genes will be needed to ascertain the genetic cause of IGHD in the remaining children.