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 the cytotoxic effect of the monoterpene indole alkaloid echitamine in-vitro and in tumour-bearing mice.

The cytotoxic effect of various concentrations of echitamine chloride was studied in HeLa, HepG2, HL60, KB and MCF-7 cell lines in-vitro and in mice bearing Ehrlich ascites carcinoma (EAC). Exposure of various cells to different concentrations of echitamine chloride resulted in a concentration-dependent cell killing, and KB cells were found to be most sensitive amongst all the cells evaluated. EAC mice treated with 1, 2, 4, 6, 8, 12 or 16 mg kg-1 echitamine chloride showed a dose-dependent elevation in the anti-tumour activity, as evident by increased number of survivors in comparison with the non-drug treated controls. The highest dose of echitamine chloride (16 mg kg-1) caused toxicity in the recipient mice, therefore 12 mg kg-1 was considered the best cytotoxic dose for its anti-tumour effect. Administration of 12 mg kg-1 echitamine chloride resulted in an increase in the median survival time (MST) up to 30.5 days, which was 11.5 days higher than the non-drug treated control (19 days). Administration of 16 mg kg-1 echitamine chloride to EAC mice resulted in a time dependent elevation in lipid peroxidation that reached a peak at 6 h post-treatment, whereas glutathione concentration declined in a time dependent manner and a maximum decline was reported at 3 h post-treatment. Our study demonstrated that echitamine chloride possessed anti-tumour activity in-vitro and in-vivo.

Molecular cloning and characterization of the macaque sperm associated antigen 9 (SPAG9): an orthologue of human SPAG9 gene.

The present study was conducted to isolate macaque proteomic homologue of human SPAG9 (EMBL nomenclature human sperm associated antigen 9: hSPAG9; Shankar et al., 1998: Biochem Biophys Res Commun 243:561-565) in order to find out whether the macaque can provide a suitable model for examining its immunocontraception effects. Macaque SPAG9 was cloned and sequenced from the macaque testis cDNA library. The macaque cDNA contained open reading frame encoding 712 amino acids. A 84.9% and 94% homology between macaque and human SPAG9 was found at protein and DNA levels. Northern analysis and RNA in situ hybridization experiments revealed testis- and stage-specific expressions of macaque SPAG9 mRNA, mainly confined to round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized native SPAG9 in macaque sperm extract in Western blotting and the acrosomal compartment region of macaque sperm in indirect immunofluorescence. Flow cytometry analysis further revealed surface localization of macaque SPAG9 in live macaque sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating macaque with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.

Isolation and characterization of a haploid germ cell specific sperm associated antigen 9 (SPAG9) from the baboon.

Previously, we cloned and sequenced a sperm specific antigen, designated as HSS (EMBL nomenclature human sperm associated antigen 9: hSPAG9) from human testis (Shankar et al.: Biochem Biophys Res Commun 243:561-565, 1998). The present study was conducted to isolate baboon proteomic homologue in order to find out whether the baboon can provide a suitable model for examining its immunocontraception effects. Baboon SPAG9 (bSPAG9) was cloned and sequenced from the baboon testis cDNA library. The baboon cDNA contained open reading frame encoding 760 amino acids. A 90.6 and 96.8% homology between baboon and human SPAG9 was found at protein and DNA levels. Analysis for tissue specificity by Northern blot procedure using various baboon tissues indicated that bSPAG9 was specifically expressed only in the baboon testis. Further, cell type expression analysis by in situ hybridization in baboon testis demonstrated the expression of bSPAG9 mRNA transcript only in the round spermatid suggesting haploid germ cell expression. Anti-human SPAG9 antibodies recognized the acrosomal compartment region of baboon sperm in indirect immunofluorescence (IIF). Flow cytometry analysis showed surface localization of bSPAG9 in live baboon sperm. The amino acid sequence data for nonhuman primate SPAG9 suggest that antibodies generated by vaccinating baboon with hSPAG9 will recognize nonhuman primate SPAG9, supporting the testing of SPAG9 contraceptive vaccine based on hSPAG9 in the nonhuman primate model.

The evaluation of the acute toxicity and long term safety of hydroalcoholic extract of Sapthaparna (Alstonia scholaris) in mice and rats.

The acute and sub-acute toxic effects of various doses of hydroalcoholic extract of Alstonia scholaris (ASE) was studied in mice and rats. The acute toxicity in mice depended on the season of collection of plant. The highest acute toxicity was observed in the ASE prepared from the summer collection followed by winter. The least toxicity was observed in the extract prepared from the bark of A. scholaris collected in the monsoon season. The administration of different doses of ASE showed a dose dependent increase in the toxicity in all species of mice. The Swiss albino mice were found to be the most sensitive followed by the DBA and C(57)BL. The crossbred mice were resistant when compared to the pure inbred strains. The oral administration of ASE was non-toxic up to a dose of 2000 mg/kg b. wt., while maximum number of animals succumbed to death after administration of 1100 mg/kg ASE by intraperitoneal route. The rats were more sensitive than the mice as the LD(50) dose of ASE was lesser for the former than the latter. The sub-acute toxicity in the rats was carried out with 120 and 240 mg/kg b. wt. ASE (1/10th and 1/5th of the LD(50) dose of ASE). The 240 mg was observed to be more toxic than 120 mg/kg ASE since it caused mortality and deformity in various organs of the recipient animals. The various biochemical parameters like AST, ALT, ACP, ALP, CK, LDH, creatinine, urea, ammonia, glucose and LPx were higher at 240 mg/kg ASE when compared with the 120 mg and the non-drug treated animals. In contrast, the total protein, albumin, DNA, RNA, cholesterol, glucose, glutathione, total thiols declined in the 240 mg/kg ASE treated animals when compared with non-drug treated controls. The hematological analysis showed a dose dependent decrease in the RBC, WBC, hemoglobin, neutrophils and monocytes, while a significant increase in the lymphocytes, eosinophils and basophils was observed. The observed toxic effect of ASE may be due to the presence of echitamine. Our studies shows that at high doses, A. scholaris exhibited marked damage to all the major organs of the body.

Influence of ginger rhizome (Zingiber officinale Rosc) on survival, glutathione and lipid peroxidation in mice after whole-body exposure to gamma radiation.

The radioprotective effect of the hydroalcoholic extract of ginger rhizome, Zingiber officinale (ZOE), was studied. Mice were given 10 mg/kg ZOE intraperitoneally once daily for five consecutive days before exposure to 6-12 Gy of gamma radiation and were monitored daily up to 30 days postirradiation for the development of symptoms of radiation sickness and mortality. Pretreatment of mice with ZOE reduced the severity of radiation sickness and the mortality at all doses. The ZOE treatment protected mice from GI syndrome as well as bone marrow syndrome. The dose reduction factor for ZOE was found to be 1.15. The optimum protective dose of 10 mg/kg ZOE was 1/50 of the LD50 (500 mg/kg). Irradiation of the animals resulted in a dose-dependent elevation in the lipid peroxidation and depletion of GSH on day 31 postirradiation; both effects were lessened by pretreatment with ZOE. ZOE also had a dose-dependent antimicrobial activity against Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and Candida albicans.