Deciphering a GPCR-lncrna-miRNA nexus: Identification of an aberrant therapeutic target in ovarian cancer.

Ovarian cancer ranks as a leading cause of mortality among gynecological malignancies, primarily due to the lack of early diagnostic tools, effective targeted therapy, and clear understanding of disease etiology. Previous studies have identified the pivotal role of Lysophosphatidic acid (LPA)-signaling in ovarian cancer pathobiology. Our earlier transcriptomic analysis identified Urothelial Carcinoma Associated-1 (UCA1) as an LPA-stimulated long non-coding RNA (lncRNA). In this study, we elucidate the tripartite interaction between LPA-signaling, UCA1, and let-7 miRNAs in ovarian cancer progression. Results show that the elevated expression of UCA1 enhances cell proliferation, invasive migration, and therapy resistance in high-grade serous ovarian carcinoma cells, whereas silencing UCA1 reverses these oncogenic phenotypes. UCA1 expression inversely correlates with survival outcomes and therapy response in ovarian cancer clinical samples, underscoring its prognostic significance. Mechanistically, UCA1 sequesters let-7 miRNAs, effectively neutralizing their tumor-suppressive functions involving key oncogenes such as Ras and c-Myc. More significantly, intratumoral delivery of UCA1-specific siRNAs inhibits the growth of cisplatin-refractory ovarian cancer xenografts, demonstrating the therapeutic potential of targeting LPAR-UCA1-let-7 axis in ovarian cancer. Thus, our results identify LPAR-UCA1-let-7 axis as a novel avenue for targeted treatment strategies.

Circulating Tumor Cell-Free DNA Genes as Prognostic Gene Signature for Platinum Resistant Ovarian Cancer Diagnosis.

Clinical management of gynecological cancer begins by optimal debulking with first-line platinum-based chemotherapy. However, in ~80% patients, ovarian cancer will recur and is lethal. Prognostic gene signature panel identifying platinum-resistance enables better patient stratification for precision therapy. Retrospectively collected serum from 11 "poor" (<6 months progression free interval [PFI]) and 22 "favorable" (>24 months PFI) prognosis patients, were evaluated using circulating cell-free DNA (cfDNA). DNA from both groups showed 50 to 10 000 bp fragments. Pairwise analysis of sequenced cfDNA from patients showed that gene dosages were higher for 29 genes and lower for 64 genes in poor than favorable prognosis patients. Gene ontology analysis of higher dose genes predominantly grouped into cytoskeletal proteins, while lower dose genes, as hydrolases and receptors. Higher dosage genes searched for cancer-relatedness in Reactome database indicated 15 genes were referenced with cancer. Among them 3 genes, TGFBR2, ZMIZ2, and NRG2, were interacting with more than 4 cancer-associated genes. Protein expression analysis of tumor samples indicated that TGFBR2 was downregulated and ZMIZ2 was upregulated in poor prognosis patients. Our results indicate that the cfDNA gene dosage combined with protein expression in tumor samples can serve as gene signature panel for prognosis determination amongst ovarian cancer patients.

Chemopreventive and Anticancer Effects of Thymoquinone: Cellular and Molecular Targets.

Thymoquinone (TQ) is a bioactive component derived from the seeds of Nigella sativa that are commonly as black cumin. Evidences indicate that the medicinal properties of TQ have been recognized for more than 2000 years. TQ has been shown to possess potent chemopreventive properties that include anti-inflammatory and anti-neoplastic activities. Recent studies have unraveled the multiple mechanisms through which TQ exerts its chemopreventive and anticancer activity in different cancer cells in a contextual manner. The present review aims to provide a brief compendium on the molecular mechanisms through which TQ inhibits signaling pathways underlying cancer genesis, progression, and metastasis.

Differential effects of thymoquinone on lysophosphatidic acid-induced oncogenic pathways in ovarian cancer cells.

Thymoquinone, a therapeutic phytochemical derived from Nigella sativa, has been shown to have a potent anticancer activity. However, it has been identified that the tumor microenvironment (TME) can attenuate the anticancer effects of thymoquinone (TQ) in ovarian cancer. Lysophosphatidic acid (LPA), a lipid growth factor present in high concentration in the TME of ovarian cancer, has been shown to regulate multiple oncogenic pathways in ovarian cancer. Taking account of the crucial role of LPA in the genesis and progression of ovarian cancer, the present study is focused on assessing the efficacy of TQ in inhibiting LPA-stimulated oncogenic pathways in ovarian cancer cells. Our results indicate that TQ is unable to attenuate LPA-stimulated proliferation or metabolic reprogramming in ovarian cancer cells. However, TQ potently inhibits the basal as well as LPA-stimulated migratory responses of the ovarian cancer cells. Furthermore, TQ abrogates the invasive migration of ovarian cancer cells induced by Gαi2, through which LPA stimulates cell migration. TQ also attenuates the activation of JNK, Src, and FAK, the downstream signaling nodes of LPA-LPAR-Gαi2 signaling pathway. In addition to establishing the differential effects of TQ in ovarian cancer cells, our results unravel the antitherapeutic role of LPA in the ovarian cancer TME could override the inhibitory effects of TQ on cell proliferation and metabolic reprogramming of ovarian cancer cells. More importantly, the concomitant finding that TQ could still sustain its inhibitory effect on LPA-stimulated invasive cell migration, points to its potential use as a response-specific therapeutic agent in ovarian cancer.

Carriers of the TCF7L2 rs7903146, rs12255372 Risk Alleles in the South Tamil Nadu T2DM Patients Present with Early Incidence and Insulin Dependence.

BACKGROUND AND AIMS: Metabolic abnormalities in T2DM (Type 2 diabetes mellitus) include classic manifestations such as impaired insulin secretion, synthesis and peripheral insulin resistance. The intronic variants rs7903146 and rs12255372 of the TCF7L2 (transcription factor 7-like 2) gene are strongly associated with risk of incidence of T2DM and impaired ß-cell functions. Studies addressing the early T2DM onset, and early insulin dependence in T2DM patients of south Tamil Nadu are still lacking, and hence the present study focuses in determining the influence of the TCF7L2 polymorphisms in the incidence and disease course in the T2DM patients of south Tamil Nadu. METHODS: Anthropometric measurements and biochemical parameters were carried out in early onset (Group A), early onset insulin dependent T2DM patients (Group B) and non-insulin dependent T2DM patients (Group C). PCR, allele specific PCR (ASP), PCR product sequencing strategies were utilized to determine the genotype and the impact of the TCF7L2 SNPs in the T2DM disease course. RESULTS: Female T2DM patients with the CT/TT rs7903146 genotype (P = 0.005) and the rs12255372 GT/TT genotype (P = 0.036) exhibited a significantly low mean age for T2DM incidence. Correlation/regression analysis in the T2DM patients revealed that rs12255372 (P = 0.042) is associated with early onset in the Group C patients and the rs7903146 (P = 0.018), rs12255372 (P = 0.026) are associated with insulin dependence in the group B patients. CONCLUSION: Screening for the TCF7L2 polymorphisms will prevent T2DM incidence and enable life style changes, appropriate therapeutic strategies that would help combat the accelerated disease course in the T2DM patients.

Reply to the Comment on "Michael Addition Based Chemodosimeter for Serum Creatinine Detection Using ( E)-3-(Pyren-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one Chalcone".

A very simple chemodosimeter has been developed for creatinine biomolecules based on Michael addition reaction by using ( E)-3-(Pyren-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2- en-1-one Chalcone. The photophysical properties of the chalcone were thoroughly analyzed using UV-vis and emission techniques. The chalcone has exhibited two absorption maxima at 297 and 407 nm which are due n-π* and π-π* transactions, respectively. This property was further confirmed by repeating UV-vis absorbance studies of the chalcones in different solvents having different polarity. The PTP chalcone has originally exhibited ICT mechanism and it is arrested while creatinine is added. However, a ratiometric response is observed due to the creatinine induced ICT mechanism and it is also clearly supported with DFT studies. In our original work, we did DFT studies for only one isomer of the creatinine. Currently, we have extended our DFT studies for another isomer also. The relative quantum yield of the PTP chalcone was calculated in sensing and standard conditions as 0.85 and 0.45, respectively.

Michael Addition Based Chemodosimeter for Serum Creatinine Detection Using ( E)-3-(Pyren-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one Chalcone.

First, a simple and highly emissive fluorescent chalcone ( E)-3-(pyren-2-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (PTP) was synthesized via simple shaking along with an excellent quantum yield of 0.85, and proved as a stable, highly sensitive, and selective biosensor for creatinine. Owing to its unique photophysical interaction with creatinine through Michael adduct formation, PTP was utilized as a Chemodosimeter for the selective recognition of creatinine in blood serum. Under optimized conditions, a broad range of creatinine detection was achieved  from 0.00000113 mg/dL to 15.8 mg/dL along with an excellent limit of detection of 0.00000065 mg/dL (0.058 nM). This biosensor is highly reproducible even for different concentration levels of creatinine. It is the very first creatinine biosensor possessing a wider linear range for clinical applications for creatinine. To ensure its clinical application, blood serum samples of people of different age groups were collected from Alpha Hospital and analyzed for creatinine by using our chemodosimeter method and compared with data obtained using a commercial method in the Alpha hospital. Our data show very good agreement with clinical data. Because clinical protocol involves trienzymes and tedious sample preparation, no doubt, our chemodosimeter will be a cheap and sensitive option compared to the existing clinical methods.

Aberrant expression of JNK-associated leucine-zipper protein, JLP, promotes accelerated growth of ovarian cancer.

Ovarian cancer is the most fatal gynecologic cancer with poor prognosis. Etiological factors underlying ovarian cancer genesis and progression are poorly understood. Previously, we have shown that JNK-associated Leucine zipper Protein (JLP), promotes oncogenic signaling. Investigating the role of JLP in ovarian cancer, our present study indicates that JLP is overexpressed in ovarian cancer tissue and ovarian cancer cells. Transient overexpression of JLP promotes proliferation and invasive migration of ovarian cancer cells. In addition, ectopic expression of JLP confers long-term survival and clonogenic potential to normal fallopian tube-derived epithelial cells. Coimmunoprecipitation and colocalization analyses demonstrate the in vivo interaction of JLP and JNK, which is stimulated by lysophosphatidic acid (LPA), an oncogenic lipid growth factor in ovarian cancer. We also show that LPA stimulates the translocation of JLP-JNK complex to the perinuclear region of SKOV3-ip cells. JLP-knockdown using shRNA abrogates LPA-stimulated activation of JNK as well as LPA-stimulated proliferation and invasive migration of SKOV3-ip cells. Studies using ovarian cancer xenograft mouse model indicate that the mice bearing JLP-silenced xenografts exhibits reduced tumor volume. Analysis of the xenograft tumor tissues indicate a reduction in the levels of JLP, JNK, phosphorylated-JNK, c-Jun and phosphorylated-c-Jun in JLP-silenced xenografts, thereby correlating the attenuated JLP-JNK signaling node with suppressed tumor growth. Thus, our results identify a critical role for JLP-signaling axis in ovarian cancer and provide evidence that targeting this signaling node could provide a new avenue for therapy.

Determinant role for the gep oncogenes, Gα12/13, in ovarian cancer cell proliferation and xenograft tumor growth.

Recent studies have shown that the gip2 and gep oncogenes defined by the α-subunits of Gi2 and G12 family of G proteins, namely Gαi2 and Gα12/13, stimulate oncogenic signaling pathways in cancer cells including those derived from ovarian cancer. However, the critical α-subunit involved in ovarian cancer growth and progression in vivo remains to be identified. Using SKOV3 cells in which the expressions of individual Gα-subunits were silenced, we demonstrate that the silencing of Gα12 and Gα13 drastically attenuated serum- or lysophosphatidic acid-stimulated proliferation. In contrast, the invasive migration of these cells were reduced only by the silencing of Gαi2 or Gα13. Analyses of the xenograft tumors derived from these Gα-silenced cells indicated that only the silencing of Gα13 drastically reduced xenograft tumor growth and prolonged the survival of the mice. Similar, but albeit reduced, effect was seen with the silencing of Gα12. On the contrary, the silencing of Gαi2 or Gαq failed to exert such effect. Thus, our studies establish for the first time that Gα12/13, the putative gep oncogenes, are the determinant α-subunits involved in ovarian cancer growth in vivo and their increased oncogenicity can be correlated with its ability to stimulate both proliferation and invasive migration.

A novel crosstalk between calcium/calmodulin kinases II and IV regulates cell proliferation in myeloid leukemia cells.

CaMKs link transient increases in intracellular Ca(2+) with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest,impairing cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27(kip1) and p16(ink4a) and reduced levels of cyclins A, B1 and D1. These findings were also confirmed in the K562 leukemic cell line. The relationship between CaMKII and CaMKIV is also observed in primary acute myeloid leukemia (AML) cells, and it correlates with their immunophenotypic profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII levels. Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to promote leukemia cell proliferation.

Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration.

Hax-1 is a multifunctional protein, which is involved in diverse cellular signaling pathways including tumor cell survival and migration. We have shown previously that cell migration stimulated by the oncogenic G protein, G13, requires Hax-1 for the formation of a functional complex involving Gα13, Rac1, and cortactin. However, the role of Hax-1 in cancer cell migration or its role in Rac1-cortactin complex formation, which is known to be required for such migration remains to be characterized. Results focused on resolving the role of Hax-1 in ovarian cancer pathophysiology indicate that Hax-1 is overexpressed in ovarian cancer cells and the silencing of Hax-1 inhibits lysophosphatidic acid (LPA)- or fetal bovine serum-stimulated migration of these cells. In addition, silencing of Hax-1 greatly reduces Rac1-cortactin interaction and their colocalization in SKOV3 cells. Mapping the structural domains of Hax-1 indicates that it interacts with cortactin via domains spanning amino acids 1 to 56 (Hax-D1) and amino acids 113 to 168 (Hax-D3). Much weaker interaction with cortactin was also observed with the region of Hax-1 spanning amino acids 169 - 224 (Hax-D4). Similar mapping of Hax-1 domains involved in Rac1 interaction indicates that it associates with Rac1 via two primary domains spanning amino acids 57 to 112 (Hax-D2) and 169 to 224 (Hax-D4). Furthermore, expression of either of these domains inhibits LPA-mediated migration of SKOV3 cells, possibly through their ability to exert competitive inhibition on endogenous Hax-1-Rac1 and/or Hax-1-cortactin interaction. More significantly, expression of Hax-D4 drastically reduces Rac1-cortactin colocalization in SKOV3 cells along with an attenuation of LPA-stimulated migration. Thus our results presented here describe for the first time that Hax-1 interaction is required for the association between Rac1 and cortactin and that these multiple interactions are required for the LPA-stimulated migration of SKOV3 ovarian cancer cells.

A novel Gfer-Drp1 link in preserving mitochondrial dynamics and function in pluripotent stem cells.

Mitochondria, the dynamic energy powerhouses of the cell, have vital roles in a multitude of cellular processes including differentiation and cell survival. Tight regulation of mitochondrial dynamics, integrity and function is indispensible for preservation of homeostasis in all cells, including pluripotent stem cells. The ability to proliferate and self-renew indefinitely bestows the pluripotent embryonic stem cells (ESCs) with immense curative potential. Mechanisms that preserve mitochondrial well-being, and therefore maintain "stemness," are vital in realizing the full potential of ESCs in therapeutic regenerative medicine. However, virtually nothing is known regarding the regulation of mitochondrial dynamics and function and the relationship thereof to overall cell fate and function in pluripotent ESCs or other somatic stem cells. Using loss- and gain-of-function approaches, we show that growth factor erv1-like (Gfer) plays an essential pro-survival role in the maintenance of murine ESC pluripotency by preserving the structural and functional integrity of their mitochondria, through modulation of the key mitochondrial fission factor Drp1.

Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.

The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.

Identification of a potent herbal molecule for the treatment of breast cancer.

BACKGROUND: Breast cancer (BCa)-related mortality still remains the second leading cause of cancer-related deaths worldwide. Patients with BCa have increasingly shown resistance and high toxicity to current chemotherapeutic drugs for which identification of novel targeted therapies are required. METHODS: To determine the effect of PDBD on BCa cells, estrogen-receptor positive (ER+)-MCF-7 and estrogen-receptor negative (ER-)-MDA 231 cells were treated with PDBD and the cell viability, apoptotic, cell cycle, Western blot and Promoter assays were performed. RESULTS: PDBD inhibits cell viability of ER+ and ER- BCa cells by inducing apoptosis without causing significant toxicity in normal breast epithelial cells. While dissecting the mechanism of action of PDBD on BCa, we found that PDBD inhibits Akt signaling and its downstream targets such as NF-kappaB activation, IAP proteins and Bcl-2 expression. On the other hand, activation of JNK/p38 MAPK-mediated pro-apoptotic signaling was observed in both ER+ and ER- BCa cells. CONCLUSION: These findings suggest that PDBD may have wide therapeutic application in the treatment of BCa.

Anticancer mechanism of plumbagin, a natural compound, on non-small cell lung cancer cells.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths in the United States. Prevailing treatment options have limited therapeutic success in lung cancer, particularly non-small cell lung cancer (NSCLC), as it becomes resistant to therapy. Hence, better therapeutic options are immediately required for lung cancer. Plumbagin, a natural compound has been recently examined for its anticancer effect on different cancers. MATERIALS AND METHODS: To determine the anticancer effect of plumbagin on NSCLC cell lines H460 and A549, cell viability, apoptotic, Western blot and reporter assays were performed. RESULTS: Plumbagin significantly inhibited the growth of H460 cells compared to A549 cells, and down-regulated the expression of EGFR/Neu and its downstream signaling (Akt, NF-kappaB, Bcl-2 and survivin) in H460 cells. In addition, plumbagin up-regulated the expression of p53 and p21(CIP1/WAF1) causing cell cycle arrest in the G2/M-phase by down-regulating G2/M regulatory proteins (cyclinB1 and Cdc25B) in H460 cells. Furthermore, it activated the JNK/p38 signaling, leading to caspase-3 activation resulting in the induction of apoptosis. CONCLUSION: Plumbagin exerted anticancer activity on NSCLC cells by modulating the pro-survival and pro-apoptotic signaling that causes induction of apoptosis.

Mithramycin analogues generated by combinatorial biosynthesis show improved bioactivity.

Plasmid pLNBIV was used to overexpress the biosynthetic pathway of nucleoside-diphosphate (NDP)-activated l-digitoxose in the mithramycin producer Streptomyces argillaceus. This led to a "flooding" of the biosynthetic pathway of the antitumor drug mithramycin (MTM) with NDP-activated deoxysugars, which do not normally occur in the pathway, and consequently to the production of the four new mithramycin derivatives 1- 4 with altered saccharide patterns. Their structures reflect that NDP sugars produced by pLNBIV, namely, l-digitoxose and its biosynthetic intermediates, influenced the glycosyl transfer to positions B, D, and E, while positions A and C remained unaffected. All four new structures have unique, previously not found sugar decoration patterns, which arise from either overcoming the substrate specificity or inhibition of certain glycosyltransferases (GTs) of the MTM pathway with the foreign NDP sugars expressed by pLNBIV. An apoptosis TUNEL (=terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay revealed that compounds 1 (demycarosyl-3D-beta- d-digitoxosyl-MTM) and 3 (deoliosyl-3C-beta- d-mycarosyl-MTM) show improved activity (64.8 +/- 2% and 50.3 +/- 2.5% induction of apoptosis, respectively) against the estrogen receptor (ER)-positive human breast cancer cell line MCF-7 compared with the parent drug MTM (37.8 +/- 2.5% induction of apoptosis). In addition, compounds 1 and 4 (3A-deolivosyl-MTM) show significant effects on the ER-negative human breast cancer cell line MDA-231 (63.6 +/- 2% and 12.6 +/- 2.5% induction of apoptosis, respectively), which is not inhibited by the parent drug MTM itself (2.6 +/- 1.5% induction of apoptosis), but for which chemotherapeutic agents are urgently needed.