Ketorolac modulates Rac-1/HIF-1α/DDX3/ß-catenin signalling via a tumor suppressor prostate apoptosis response-4 (Par-4) in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most difficult-to-treat form of kidney cancer with a median 5-year survival of 10% under metastatic setting. In RCC, although cytoreductive nephrectomy is common, approximately 20-30% of patients will develop recurrent cancer after surgery, which highlights the need for an effective therapy. Rho-GTPases viz, Rac-1 and Cdc42 are the central regulators of cancer cell migration and invasion and thus metastasis in multiple cancer types. Hence, we elucidated the role of Ketorolac, a modulator Rho-GTPases against RCC through potentiation of tumor suppressor Par-4. The effect of Ketorolac alone and in combination on proliferation, apoptosis, cell-cycle progression, migration, tumor inhibition and their related markers were studied. Moreover, Ketorolac's impact on metastasis by influencing Rac-1/HIF-1α/DDX3/ß-catenin signalling was studied with respect to its ability to modulate the expression of tumor suppressor Par-4, and this mechanism was confirmed by siRNA knockdown studies. Ketorolac induced cytotoxicity in a panel of renal cells including patient derived tumor cells with IC50 2.8 to 9.02 mM and 0.28 to 3.8 mM in monolayer and anchorage independent clonogenic assays respectively. Ketorolac caused significant down regulation of proliferation (Ki-67, Cyclin D1, pRB and DDX3), migration/invasion (Rac-1, Cdc42, and Tiam1), and angiogenesis (HIF-1α and VEGF) markers as studied by gene and protein expression. Moreover, it caused a significant upregulation of tumor suppressor Par-4 known to be downregulated in RCC. This mechanism was further confirmed by using siRNA knockdown studies where we could demonstrate a negative relation between the expression of Par-4 and Rac-1/Cdc42. Importantly, Ketorolac alone and in combination with Sunitinib showed tumor growth inhibition (TGI) of 73% and 86% respectively in xenograft model. This anti-tumor activity was further corroborated by down regulation of Rac-1/Cdc42/HIF-1α/DDX3/ß-catenin signalling. This is the first report which implicates the role of Ketorolac against RCC by acting as a small molecule secretagogue causing upregulation of Par-4 in autocrine and paracrine manner. Consequently, these findings suggest that Par-4 can serve as a valuable therapeutic target and a prognostic marker for the treatment of RCC.

Prostate Apoptosis Response-4 (Par-4): A Novel Target in Pyronaridine-Induced Apoptosis in Glioblastoma (GBM) Cells.

Glioblastoma (GBM) is an aggressive form of brain tumor with a median survival of approximately 12 months. With no new drugs in the last few decades and limited success in clinics for known therapies, drug repurposing is an attractive choice for its treatment. Here, we examined the efficacy of pyronaridine (PYR), an anti-malarial drug in GBM cells. PYR induced anti-proliferative activity in GBM cells with IC50 ranging from 1.16 to 6.82 µM. Synergistic activity was observed when PYR was combined with Doxorubicin and Ritonavir. Mechanistically, PYR triggered mitochondrial membrane depolarization and enhanced the ROS levels causing caspase-3 mediated apoptosis. PYR significantly decreased markers associated with proliferation, EMT, hypoxia, and stemness and upregulated the expression of E-cadherin. Interestingly, PYR induced the expression of intracellular as well as secretory Par-4, a tumor suppressor in GBM cells, which was confirmed using siRNA. Notably, Par-4 levels in plasma samples of GBM patients were significantly lower than normal healthy volunteers. Thus, our study demonstrates for the first time that PYR can be repurposed against GBM with a novel mechanism of action involving Par-4. Herewith, we discuss the role of upregulated Par-4 in a highly interconnected signaling network thereby advocating its importance as a therapeutic target.

Association of AMH and AMHR2 gene polymorphisms with ovarian response and pregnancy outcomes in Indian women.

PURPOSE: To evaluate the association of single-nucleotide polymorphisms (SNPs) in the anti-Müllerian hormone (AMH) and AMH type II receptor (AMHR2) genes with ovarian response and clinical pregnancy outcomes in women undergoing controlled ovarian hyperstimulation. METHODS: In this prospective study, we genotyped AMH polymorphisms (c. -649 T > C, c. 146 T > G, c. 252 G > A, and c. 303 G > A) in 365 women and AMHR2 polymorphisms (c. -482 A > G, c. 622-6 C > T, c. 4952 G > A, c. 10 A > G) in 80 women undergoing controlled ovarian hyperstimulation for IVF. RESULTS: Higher doses of exogenous FSH and lower numbers of preovulatory follicles were noted in women having AMH c. -649 T > C and AMH c. -146 T > G polymorphisms, respectively. Overall, we found that the presence of a polymorphic genotype (homozygous or heterozygous) at positions c. -649 T > C, c. 146 T > G, c. 252 G > A, and c. 303 G > A in the AMH gene was associated with higher doses of FSH for ovulation induction (p < 0.001). Interestingly, a higher live birth rate was noted in women with a homozygous polymorphic genotype for all four AMH SNPs investigated while none of the women showing a homozygous polymorphic genotype at all AMHR2 SNPs investigated in this study had a live birth. CONCLUSION: Our results show that presence of AMHR2 SNPs (c. 482 A > G, c. 622-6 C > T, c. 4952 G > A, and c. 10 A > G) negatively correlate with live birth rate. However, these findings need to be validated by using larger sample size.

Risk association of BST2 gene variants with disease progression in HIV-1 infected Indian cohort.

Bone marrow stromal cell antigen 2 (BST2) is an interferon induced host restriction factor for HIV-1 that blocks the release of nascent virions from infected T cells. We aimed to characterize BST2 gene variants in HIV-1 positive individuals in Indian cohort and study the association of these variants with disease progression in long term non progressors (LTNPs) and progressors. Archived samples of 32 LTNPs, 17 progressors, and 78 controls were screened for BST2 gene polymorphisms using Sanger's sequencing method. Frequency distribution, survival analysis and bioinformatics tools were used to study the association of BST2 variants with disease progression. Eighteen variants of BST2 gene were observed in Indian cohort. Intronic SNP rs919267T/C (OR = 4.489 [0.8494-27.03], p = .04157) and exonic SNP rs13485C/G (OR = 3.887 [0.8262-25.56], p = .0488) were found to be significantly associated with disease progression. Also, rs13485C/C genotype in combination with rs919267C/T (OR = 9.406 [1.384-111], p = .0085) and rs145303329 Δ19bp (OR = 3.887 [0.826-25.5], p = .048) were found to be significantly associated with disease progression. 19 bp indel rs145303329 and its allele c.1-443_1-442insCGCCCCCAGAC[C/T]CAGGCCC from BST2 promoter also showed association with disease progression (OR = 12.97 [0.9731-850.5], p = .026). Docking of AP2 repressor with above allele showed the total binding energy of LTNPs and progressors to be -2581.42 kcal/mol and -3563.27/-3562.84 kcal/mol respectively. We have identified the novel association of three BST2 gene SNPs; rs919267, rs13485 and indel rs145303329 from Indian cohort to be associated with the risk of HIV-1 disease progression for the first time.

Functional characterization of two naturally occurring mutations V221G and T449N in the follicle stimulating hormone receptor.

Naturally occurring mutations in follicle stimulating hormone receptor (FSHR) affect the receptor function. Here, we characterized two such previously reported mutations, V221G and T449N, in the extracellular domain and transmembrane helix 3, of FSHR, respectively. Functional studies with the V221G mutant demonstrated an impairment in FSH binding and signaling. Validation of X-ray crystallography data indicating the contribution of FSHR specific residues in the vicinity of V221 to contribute to FSH-FSHR interaction was carried out. In vitro mutational studies showed that these residues are determinants of both FSH binding and FSH induced signaling. Analysis of the T449N mutation revealed that it results in an increase in FSH binding and high cAMP response at lower doses of FSH. A marginal hCG induced and no TSH induced cAMP production was also observed. These findings corroborated with the clinical manifestations of primary amenorrhea (V221G) and spontaneous ovarian hyperstimulation syndrome (T449N) in women harbouring these mutations.

The rs10993994 in the proximal MSMB promoter region is a functional polymorphism in Asian Indian subjects.

BACKGROUND: The microseminoprotein gene encoding prostate secretory protein of 94 amino acids (PSP94) harbours a potential risk allele (rs10993994) for prostate cancer (PCa) in its promoter region. However, studies on rs10993994 have been sparse in Asian Indians. METHODS: The present study recruited a sample population of 44 benign prostatic hyperplasia patients, 33 PCa patients and 60 healthy participants, of which, participants without other confounding risk factors for PCa were retained. The serum PSP94 (sPSP94) levels were measured by a serum-based ELISA in an earlier study. A novel RFLP technique was developed to screen for rs10993994 which was validated with direct sequencing. RESULTS: Sequencing showed additional 4 SNPs (rs41274660, rs141211965, rs12770171, rs10669586) and 2 novel variants (GenBank accession nos. KM265191 and KM265192). In silico DNA topographical studies predicted that KM265192 would have higher cleavage intensity and more accessibility for binding of transcription factors. Even though, similar frequencies were observed for all the variants in all the three study groups, the risk allele 'T' (rs10993994) was seen to be associated with reduced PSP94 expression both at mRNA and protein level. Further, mRNA expression as studied by real-time PCR correlated positively with sPSP94 levels. Interestingly, CC genotype of rs10993994 showed highest sPSP94 levels in all the three study groups and was associated with Gleason score ≤7 in PCa patients. In contrast, TT genotype of rs10993994 was associated with lesser sPSP94 levels and with aggressiveness of PCa. CONCLUSION: rs10993994 was found to be a functional SNP in the studied Asian Indian population.

Functional characterization of two naturally occurring mutations (Val514Ala and Ala575Val) in follicle-stimulating hormone receptor.

CONTEXT: Inactivating mutations have been reported in subjects with primary/secondary amenorrhea, whereas activating mutations are rare and seen only in women with ovarian hyperstimulation syndrome (OHSS). In the present study, we describe the functional characterization of the two mutations Val(514)Ala (novel mutation) and Ala(575)Val in FSH receptor (FSHR) identified in women with OHSS developed during in vitro fertilization and primary amenorrhea, respectively. OBJECTIVE: The objective of the investigation was to study the effect of mutations (514 and 575) on FSHR activity by in vitro functional studies. SETTING: The study was conducted at an academic research institute and a private in vitro fertilization clinic. METHODS: The site-directed mutagenesis was carried out to generate the mutations at position 514 and 575 in pSG5-FSHR construct. Stable cell lines expressing wild type or each of the mutant receptor were generated using Chinese hamster ovary cells. Functional characteristics of both the mutant receptors were assessed by a radioreceptor assay and a cAMP assay. RESULTS: The mutant receptor 514 showed increased cell surface expression as compared with the wild-type (WT) receptor. Although the hormone binding characteristics were similar to the WT receptor, its signaling activity was distinctly higher at lower dose of FSH as monitored by a cAMP assay. On the other hand, the mutant receptor 575 showed lower cell surface expression and higher internalized hormone receptor complex. Additionally, a dose-dependent increase in the cAMP accumulation was not observed in the case of this mutant as compared with WT. CONCLUSION: OHSS and primary amenorrhea observed in the two affected women, respectively, could be attributed to the functional characteristics of respective mutant FSHR.

Novel FSH receptor mutation in a case of spontaneous ovarian hyperstimulation syndrome with successful pregnancy outcome.

The objective is to study the FSH receptor (FSHR) for mutations in a case of spontaneous ovarian hyperstimulation syndrome (sOHSS). This is a single case study and it examined patient who presented with spontaneous critical OHSS in early pregnancy and had successful good obstetric outcome. Intervention of this study was analysis of blood for genetic analysis of FSHR postdelivery. The main outcome measure noted was FSHR mutation. The study resulted in a novel, here though unreported, heterozygous mutation in FSHR gene at nucleotide position 1346 (AC(1346)T to AAT) in exon 10 yielding a threonine to asparagine (Thr(449)Asn) substitution in the transmembrane domain helix 3 of the FSHR. To conclude FSHR gene analysis can add to our understanding of sOHSS.

Association of allelic combinations of FSHR gene polymorphisms with ovarian response.

During an IVF protocol, exogenous FSH is administered to women for ovulation induction. The ovarian response to gonadotrophin stimulation is variable and unpredictable in these women. The FSHR is the most studied gene in relation to ovarian response. The association of a FSHR gene polymorphism at position 680 (p.Asn680Ser) with ovarian response has been well documented. Recently, a polymorphism at position -29 in the 5'-untranslated region of FSHR (g.-29G>A) has been reported to be associated with poor ovarian response and reduced FSHR expression. The present study evaluated the combined effect of the polymorphisms at positions -29 and 680 of FSHR with type of ovarian response and receptor expression. The two FSHR gene polymorphisms together formed four discrete haplotypes and nine allelic combinations. Various clinical parameters revealed that 75% of the subjects with A/A-Asn/Asn genotype were poor ovarian responders (odds ratio 7.92; P=0.009). The relative FSHR mRNA expression in granulosa cells indicated that subjects with A/A-Asn/Asn genotype express significantly lower level of FSHR as compared to the subjects with G/G-Asn/Ser genotype (P=0.029). These results indicate that A/A-Asn/Asn genotype could be used as a potential marker to predict poor ovarian response. The action of FSH is mediated by its receptor (FSHR) present on the granulosa cells in the ovary. Any alterations in the hormone or its receptor are likely to disrupt its normal function, thus causing infertility. Several alterations (mutations/polymorphisms) of the FSHR gene have been reported in women with primary or secondary amenorrhoea. It has also been reported that FSHR gene polymorphisms are associated with variable ovarian response to FSH stimulation during IVF. Women may show poor or normal or hyperovarian response to FSH stimulation. It is well documented that the level of FSHR expression has a great effect on FSH action and is associated with ovarian response. In the present study, we screened normally menstruating women undergoing IVF due to tubal/male factor or unexplained infertility. We analysed two polymorphisms of FSHR, g-29G>A and p.Asn680Ser, in these women. In the subjects studied, 75% women with A/A-Asn/Asn genotype were observed to be poor ovarian responders to FSH stimulation. FSHR expression at the transcript level was observed to be significantly lower in women with A/A-Asn/Asn genotype as compared to women with G/G-Asn/Ser genotype. We also observed that women with A/A-Ser/Ser genotype were not present in the study population. These findings indicate the significance of A/A-Asn/Asn genotype as a predictive marker for poor ovarian response to FSH stimulation.

Follicle-stimulating hormone receptor polymorphism (G-29A) is associated with altered level of receptor expression in Granulosa cells.

CONTEXT: Polymorphisms of the FSHR gene are associated with variable ovarian response to FSH stimulation in subjects undergoing in vitro fertilization (IVF) treatment. The type of ovarian response is correlated with the level of FSH receptor (FSHR) expression on granulosa cells. OBJECTIVE: We investigated whether the polymorphism at position -29 in the promoter of the FSHR gene may contribute in altered receptor expression. DESIGN AND PATIENTS: FSHR polymorphism at position -29 was studied in 100 subjects undergoing IVF treatment. Association of this polymorphism with level of FSHR expression was retrospectively analyzed. SETTING: The study was conducted at an academic research institute and private IVF clinic. METHODS: The genotype at position -29 of the FSHR gene was studied in IVF subjects by PCR-restriction fragment length polymorphism. Total RNA and protein was extracted from granulosa cells. The relative FSHR mRNA expression was carried out by real-time PCR. The receptor protein expression was evaluated by Western blot and confocal microscopy. RESULTS: The clinical and endocrinological parameters revealed that almost 72% of subjects with the AA genotype at position -29 of FSHR gene were poor ovarian responders (odds ratio 8.63, 95% confidential interval 1.84-45.79; P = 0.001). The lower cleavage intensity predicted by in silico analysis for A allele as compared with the G allele suggest the difference in the DNA-protein binding affinity. The relative expression of FSHR at mRNA and protein level was significantly reduced in subjects with AA genotype as compared with the GG genotype. CONCLUSION: Poor ovarian response observed in subjects with the AA genotype at position -29 of the FSHR gene is due to reduced receptor expression.

Follicle stimulating hormone receptor gene variants in women with primary and secondary amenorrhea.

PURPOSE: This retrospective study was designed to analyze the FSHR gene variants in subjects with primary and secondary amenorrhea with hypergonadotropic hypogonadism. MATERIALS AND METHODS: Eighty six women with primary or secondary amenorrhea and 100 normally cycling proven fertile women of Indian origin were retrospectively studied. These subjects were systematically screened for entire FSHR gene. RESULTS: The frequency distribution of polymorphism at -29 position of FSHR gene is altered in women with primary and secondary amenorrhea as compared to controls. AA genotype at -29 position of FSHR gene seems to be associated with increased serum FSH levels in the study subjects. We have identified a novel homozygous mutation C(1723)T (Ala(575)Val) in one woman with primary amenorrhea. CONCLUSIONS: Our findings suggest that increased serum FSH levels in subjects with primary amenorrhea correlated to FSHR genotype at position -29. We identified a novel homozygous mutation C(1723)T (Ala(575)Val) in a woman with primary amenorrhea.

Poor ovarian response to gonadotrophin stimulation is associated with FSH receptor polymorphism.

Similarities in the phenotype observed in women with FSH receptor mutation and in FSH receptor knockout mice have clearly established a critical role of this protein in normal gonadal function. Two common single nucleotide polymorphisms in the exonic region of the FSH receptor gene have been shown to be associated with altered ovarian response in subjects undergoing gonadotrophin treatment. Recent in-vitro studies have shown that the A allele at the -29 position in the 5 untranslated region of the FSH receptor gene is associated with impaired transcriptional activity. Differential expression of the FSH receptor and its function may be one of the factors responsible for altered ovarian response. These observations prompted a study of the association between FSH receptor genotype at the -29 position and ovarian response in women undergoing gonadotrophin treatment. Analysis of the data revealed that the subjects with AA genotype at the -29 position required the highest amount of exogenous FSH for ovulation induction, and oestradiol concentrations before the day of human chorionic gonadotrophin administration were significantly lower (P = 0.015) compared with the GA genotype. The number of pre-ovulatory follicles and retrieved oocytes were lowest in the subjects with AA genotype. These results indicate that the AA genotype at position -29 may be associated with the poor ovarian response.

Follicle-stimulating hormone receptor polymorphism (Thr307Ala) is associated with variable ovarian response and ovarian hyperstimulation syndrome in Indian women.

OBJECTIVE: To evaluate the association of FSH receptor polymorphism and ovarian response. DESIGN: Retrospective study. SETTING: Academic research institute and private IVF clinic. PATIENT(S): Fifty women were recruited in an assisted reproductive technology program (ART) and 100 proven fertile women of Indian origin. INTERVENTION(S): Polymerase chain reaction, restriction fragment-length polymorphism for detecting polymorphisms at T(307)A and N(680)S. MAIN OUTCOME MEASURE(S): FSH receptor polymorphisms, serum FSH, and estradiol levels, amount of FSH administered, occurrence of ovarian hyperstimulation syndrome (OHSS). RESULT(S): Prevalence of polymorphism at 307 position was 24%, 53%, and 23% in controls and 24%, 62%, and 14% in ART subjects for TT, TA, and AA, respectively, whereas at position 680, it was 31%, 56%, and 13% in controls and 42%, 46%, and 12% in ART subjects for NN, NS, and SS, respectively. The amount of FSH required for ovulation induction was low in AA compared with TT and TA subjects; the estradiol levels before and on the day of hCG administration were significantly higher. Eighty-five percent of the subjects with AA genotype developed OHSS. CONCLUSION(S): In Indian women, the subjects with AA genotype require low amounts of FSH for ovarian stimulation and have an increased risk of developing OHSS.