Abdul S. Ansari

Sperm characteristics and ultrastructure of testes of rats after long-term treatment with the methanol subfraction of Carica papaya seeds

The contraceptive efficacy of Carica papaya seeds after short-term evaluation has been well established. We have examined the safety and mechanism of contraception in rats after long-term treatment with the methanol subfraction (MSF) of C. papaya seeds. The test substance was administered orally to the male albino rats (n = 40) at 50 mg per kg body weight each day for 360 days. Control animals (n = 40) received olive oil as a vehicle. Recovery was assessed up to 120 days after treatment withdrawal. Sperm parameters, serum testosterone levels, fertility, histology and ultrastructure of the testis, haematology and serum clinical chemistry were evaluated to establish the safety and efficacy of the test substance. Safety of long-term treatment was evidenced by unaltered health status, organ weight, haematology and clinical chemistry, and by an increase in body weight. The mechanism of contraception was shown by reduction in nuclear and cytoplasmic volume, normal nuclear characteristics and vacuolization in the cytoplasmic organelles of the Sertoli cells, as well as nuclear degeneration in spermatocytes and spermatids indicating disturbed spermatogenesis. Leydig cells were normal. Initial effects were observed in Sertoli cells at 60 days of treatment. Spermatocytes and spermatids were affected after 120-240 days of treatment. A significant decline in sperm count and viability, total inhibition of sperm motility, increased numbers of sperm abnormalities, normal serum testosterone levels and 100% sterility were evident after 60 days of treatment. All the altered parameters, including percent fertility, were restored to control level 120 days after treatment withdrawal. It is concluded that the MSF is safe for long-term treatment and the mechanism of contraception is shown by its effect on spermatid differentiation in the testis, possibly mediated by the Sertoli cell factors.

Safety evaluation of long term oral treatment of methanol sub-fraction of the seeds of Carica papaya as a male contraceptive in albino rats.

ETHNOPHARMACOLOGICAL RELEVANCE The manuscript is one of the series of attempts in authenticating scientific documentation of the seeds of Carica papaya being traditionally used for contraception. AIMS OF THE STUDY To establish safety of the methanol sub-fraction (MSF) of the seeds of Carica papaya as a male contraceptive following long term oral treatment. MATERIALS AND METHODS MSF was administered orally to albino rats at multiples of contraceptive dose (CD) at 50 (1x), 100 (2x), 250 (5x) and 500 (10x)mg/kg body weight daily for 52 weeks. Body weight, organs weight, morbidity, mortality, clinical chemistry, sperm analysis, histopathology and serum testosterone were evaluated to assess the safety and contraceptive efficacy. RESULTS MSF treatment at various dose regimens, daily for 52 weeks did not show significant changes in body weight, organs weight, food and water intake and pre-terminal deaths compared to those of control animals. Sperm count and viability in 50mg/kg body weight treated animals and the weight of epididymis, seminal vesicle and prostate of all the treated animals showed significant reduction compared to control. Cauda epididymal spermatozoa of 50mg/kg body weight treated animals were immotile. Azoospermia was observed in 100, 250 and 500 mg/kg body weight treated animals. Serum clinical parameters, serum testosterone and histopathology of vital organs were comparable to those of control animals. Histology of testis revealed adverse effects on the process of spermatogenesis, while the histology of epididymis, seminal vesicles and ventral prostate showed no changes compared to control. CONCLUSION The long term daily oral administration of MSF affects sperm parameters without adverse side effects and is clinically safe as a male contraceptive.

The novel cancer-testis antigen A-kinase anchor protein 4 (AKAP4) is a potential target for immunotherapy of ovarian serous carcinoma

Ovarian cancer is one of the neoplasms affecting the reproductive tract associated with high mortality rate because of limited therapeutic options and an elevated incidence of chemoresistance and recurrence. In this context, immunotherapy may constitute a promising approach to improve survival rates and clinical outcome, raising the need for specific target antigens. Cancer-testis antigens (CTAs) are considered promising candidates in this sense because they are aberrant expressed by various malignancies but not by non-transformed tissue, with the exception of testes. Here, we examined the expression and potential to promote humoral immune responses of a novel CTA, A-kinase anchor protein 4 (AKAP4), among 38 ovarian carcinoma patients. Our results reveal that AKAP4 was expressed at both the mRNA and protein levels in 89% (34/38) of ovarian carcinoma tissue specimens but not in 21 matched adjacent non-cancerous tissues. In addition, a humoral response against AKAP4 was detected in 58% (22/38) of ovarian carcinoma patients by ELISA. In particular, 65% (22/34) patients bearing an AKAP4-expressing tumor exhibited circulating anti-AKAP4 antibodies. Interestingly, the majority of specimens were categorized as ovarian serous adenocarcinoma and serous papillary carcinoma, of which 93% (28/30) and 100% (6/6), respectively, expressed AKAP4. A humoral response against AKAP4 was detected in 79% (19/24) and 67% (4/6) of ovarian serous adenocarcinoma and serous papillary carcinoma patients, respectively. The presence of circulating anti-AKAP4 antibodies suggests the AKAP4 is highly immunogenic in ovarian serous carcinoma patients. Our study lays the foundations for exploring AKAP4 as a potential target for the immunotherapy of ovarian cancer.

Sperm characteristics and teratology in rats following vas deferens occlusion with RISUG and its reversal

The functional success of the reversal of vas occlusion by styrene maleic anhydride (RISUG), using the solvent vehicle, Dimethyl Sulphoxide (DMSO), has been investigated. Reversal with DMSO was carried out in Wistar albino rats 90 days after bilateral vas occlusion. The body weight, organ weight, sperm characteristics, fertility test and teratology, including skeletal morphology were evaluated in vas occlusion and reversal animals and in F(1) progenies to assess the functional success of the occlusion and reversal. Body weight, organ weight and the cauda epididymal sperm characteristics of vas occlusion and reversal animals and of F(1) progenies were comparable to control. Ejaculated spermatozoa in the vaginal smear showed detached head/tail, acrosomal damage, bent midpiece, bent tail and morphological aberrations in sperm head after vas occlusion, which returned to normal, 90 days after reversal. Monthly fertility test, post-injection showed 0% fertility, which improved gradually and 100% fertility was achieved 90 days after reversal. The fertility/pregnancy/implantation record and skeletal morphology of the offspring were comparable to control. The results suggest functional success and safety of vas occlusion reversal by DMSO.

Down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells: possible implications in targeted therapy

BackgroundRecently, we reported an association of a novel cancer testis (CT) antigen, sperm-associated antigen 9 (SPAG9) expression in breast cancer clinical samples, indicating its potential role in carcinogenesis. Around 15% breast cancers are designated as triple-negative for which treatment modalities are limited. Therefore, in the present study, we assessed the role of SPAG9 in triple-negative breast cancer cells.MethodsSPAG9 mRNA and protein expression was investigated in various breast cancer cells of different hormone receptor status and different subtypes by employing reverse transcriptase-polymerase chain reaction (RT-PCR), real time PCR, Western blotting, indirect immunofluorescence (IIF) and fluorescence activated cell sorting (FACS). Employing plasmid-based small interfering RNA (siRNA) approach, knockdown of SPAG9 was carried out in triple-negative breast cancer cells, MDA-MB-231, to assess its role on various malignant properties in vitro and in vivo.ResultsSPAG9 mRNA and protein expression was detected in all breast cancer cells. Further, IIF results showed that SPAG9 was predominantly localized in the cytoplasm of breast cancer cells. FACS analysis revealed distinct SPAG9 surface localization in breast cancer cells. Gene silencing of SPAG9 resulted in significant reduction in cellular proliferation, colony forming ability, migration, invasion and cellular motility of MDA-MB-231 cells. Further, ablation of SPAG9 expression resulted in reduction in the tumor growth of human breast cancer xenograft in nude mice in vivo.ConclusionsIn summary, our data indicated that down regulation of SPAG9 reduces growth and invasive potential of triple-negative breast cancer cells, suggesting that SPAG9 may be a potential target for therapeutic use.

Expression and humoral response of A-kinase anchor protein 4 in cervical cancer.

Background Cervical cancer is one of the major gynecologic cancers. In developing countries, because of a lack of medical support and infrastructure, cervical cancer is the leading cause of cancer-related deaths. Therefore, there is a need to identify novel biomarkers for cervical cancers. In this context, cancer-testis (CT) antigens represent a unique class of tumor antigens that have been shown to be associated with various solid tumors. These antigens have restricted expression in testis and no expression in somatic tissues. Because of their restricted expression, CT antigens are novel candidate molecules for early detection and diagnosis and immunotherapy. In the present study, novel CT antigen A-kinase anchor protein 4 (AKAP4) expression and humoral response were investigated in patients with cervical cancer. Methods In this study, 74 cervical cancer tissue specimens, which included different tumor stages (stage I [n = 35], stage II [n = 39]) and histologic grades (grade 1 [n = 17], grade 2 [n = 46], and grade 3[n = 11]) and 62 adjacent noncancerous tissue specimens were investigated for AKAP4 gene expression by using reverse transcriptase polymerase chain reaction and in situ RNA hybridization. Furthermore, AKAP4 protein expression was determined by immunohistochemistry. In addition, humoral response against purified recombinant AKAP4 protein was determined in available sera of 70 patients with cervical cancer by enzyme-linked immuno assay (ELISA). Findings Our study revealed that AKAP4 gene and protein expression was detected in 86% of total patients with cervical cancer. Based on the AKAP4 immunoreactivity score, most of stage I (n = 22/29) and stage II (n = 30/35) specimens revealed high AKAP4 expression (≥50% AKAP4-positive cells). A-kinase anchor protein 4 expression was significantly associated with early grades tumor specimens (P = 0.023). In addition, humoral response was detected in 53% of patients irrespective of stages, lymph node positivity, and grades. Conclusions Collectively, our data indicate the putative role of AKAP4 in early tumorigenesis and may be implicated as a biomarker and immunotherapeutic target for cervical cancer.

Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer

BackgroundBreast cancer is one of the leading cause of cancer-related deaths in women worldwide and increasing rapidly in developing countries. In the present study, we investigated the potential role and association of HSP70-2 with breast cancer.MethodsHSP70-2 expression was examined in 154 tumor and 103 adjacent non-cancerous tissue (ANCT) specimens and breast cancer cell lines (MCF7, BT-474, SK-BR-3 and MDA-MB-231) by RT-PCR, quantitative-PCR, immunohistochemistry, Western blotting, flow cytometry and indirect immunofluorescence. Plasmid driven short hairpin RNA approach was employed to validate the role of HSP70-2 in cellular proliferation, senescence, migration, invasion and tumor growth. Further, we studied the effect of HSP70-2 protein ablation on signaling cascades involved in apoptosis, cell cycle and Epithelial-Mesenchymal-Transition both in culture as well as in-vivo human breast xenograft mouse model.ResultsHSP70-2 expression was detected in majority of breast cancer patients (83 %) irrespective of various histotypes, stages and grades. HSP70-2 expression was also observed in all breast cancer cells (BT-474, MCF7, MDA-MB-231 and SK-BR-3) used in this study. Depletion of HSP70-2 in MDA-MB-231 and MCF7 cells resulted in a significant reduction in cellular growth, motility, onset of apoptosis, senescence, cell cycle arrest as well as reduction of tumor growth in the xenograft model. At molecular level, down-regulation of HSP70-2 resulted in reduced expression of cyclins, cyclin dependent kinases, anti-apoptotic molecules and mesenchymal markers and enhanced expression of CDK inhibitors, caspases, pro-apoptotic molecules and epithelial markers.ConclusionsHSP70-2 is over expressed in breast cancer patients and was involved in malignant properties of breast cancer. This suggests HSP70-2 may be potential candidate molecule for development of better breast cancer treatment.

Gene silencing of A-kinase anchor protein 4 inhibits cervical cancer growth in vitro and in vivo

Earlier, we reported an association of A-kinase anchor protein 4 (AKAP4) expression in cervical cancer patient specimens, indicating its implications as an immunotherapeutic target. In this study, we investigated the possible role of AKAP4 in cervical carcinogenesis. AKAP4 messenger RNA and protein expression was assessed in four cervical cancer cell line models, C-33A, CaSki, HeLa and SiHa. Gene silencing approach was employed to investigate the potential role of AKAP4 in cellular growth, proliferation, colony-forming ability, migration and invasion in aggressive squamous cell carcinoma cells (SiHa). Further, the effect of downregulation of AKAP4 on tumor growth was examined in the cervical cancer xenograft model in nude mice. Our data clearly indicated that AKAP4 was expressed in all cervical cancer cells at the gene and protein level. We also observed distinct cytoplasmic and surface localization by indirect immunofluorescence and flow cytometry, respectively. Ablation of AKAP4 protein caused significant inhibition in cellular proliferation, colony-forming ability, migration and invasion ability of SiHa cells. Further, gene silencing of AKAP4 also resulted in reduced tumor growth in nude mice in vivo. Collectively, AKAP4 surface localization and its significant association with malignant properties of cervical cancer cells imply its clinical utility as an immunotherapeutic target.

Evaluation of efficacy and safety of recombinant sperm-specific contraceptive vaccine in albino mice.

This study was undertaken to evaluate the efficacy and safety of recombinant SP‐10 protein for male contraception.

Sperm associated antigen 9 (SPAG9) expression and humoral response in benign and malignant salivary gland tumors

Salivary gland cancers are highly aggressive epithelial tumor associated with metastatic potential and high mortality. The tumors are biologically diverse and are of various histotypes. Besides, the detection and diagnosis is a major problem of salivary gland cancer for available treatment modalities. In the present study, we have investigated the association of sperm associated antigen 9 (SPAG9) expression with salivary gland tumor (SGT). Clinical specimens of benign (n = 16) and malignant tumors (n = 86) were examined for the SPAG9 expression. In addition, the sera and adjacent non-cancerous tissues (n = 72) from available patients were obtained. Our in situ RNA hybridization and immunohistochemistry (IHC) analysis revealed significant difference (p = 0.0001) in SPAG9 gene and protein expression in benign (63%) and malignant tumor (84%) specimens. Further, significant association was also observed between SPAG9 expression and malignant tumors (P = 0.05). A cut-off value of >10% cells expressing SPAG9 protein designated as positive in IHC, predicted presence of malignant SGT with 83.72% sensitivity, 100% specificity, 100% PPV and 83.72% NPV. Humoral response against SPAG9 protein was generated in 68% of SGT patients. A cut-off value of 0.212 OD for anti-SPAG9 antibodies in ELISA predicted presence of malignant SGT with 69.23% sensitivity, 100% specificity, 100% PPV and 78.94% NPV. Collectively, our data suggests that the majority of SGT show significant difference and association among benign and malignant tumors for SPAG9 gene and protein expression and also exhibit humoral response against SPAG9 protein. Hence, SPAG9 may be developed as a biomarker for detection and diagnosis of salivary gland tumors.