Nirmal K. Lohiya

Sperm-Associated Antigen 9 Is Associated With Tumor Growth, Migration, and Invasion in Renal Cell Carcinoma

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiation and chemotherapy. Current therapies for RCC patients are inefficient due to the lack of diagnostic and therapeutic markers. Our recent studies have suggested an association of sperm-associated antigen 9 (SPAG9) with ovarian carcinomas. In the present study, we investigated the clinical relevance of SPAG9 in RCC patients. RT-PCR analysis showed expression of SPAG9 transcript in RCC tissues and RCC cell lines. In situ RNA hybridization and immunohistochemistry analyses confirmed the expression of SPAG9 in 88% of cancer patients, suggesting that SPAG9 participates in renal cancer. In addition, immunoblotting and ELISA analyses revealed a humoral immune response against SPAG9 in the sera of RCC patients but not in healthy individuals. Consistent with the clinical findings, knockdown of SPAG9 expression in RCC cells with specific siRNA significantly reduced cell growth and colony formation. Using in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also significantly inhibited. Furthermore, in vivo xenograft studies in nude mice revealed that administration of a SPAG9 siRNA plasmid significantly inhibited tumor growth. In conclusion, SPAG9 expression is associated with clinicopathologic features of tumors, suggesting that SPAG9 could contribute to the early spread of cancer. These results indicate that SPAG9 may have a role in tumor development and metastasis and thus could serve as a novel target for early detection and treatment of RCC.

Reversible contraception with chloroform extract of Carica papaya Linn. seeds in male rabbits.

The contraceptive efficacy and reversibility of the chloroform extract of the seeds of Carica papaya in adult male rabbits were investigated. Eighteen adult male rabbits were divided into three groups of six animals each; Group I--control, Group II--administered chloroform extract of the seeds of Carica papaya at 20 mg/animal/d for 150 d by gavage, and Group III--administered the seed extract at 50 mg/animal/d for 150 d. Body weight and organ weight, semen analysis, sperm morphology by scanning electron microscopy, semen biochemistry, histology of the testis, haematology, serum clinical biochemistry, and the fertility status of the control and the treated animals were evaluated. Body weight and the weight of the testis, epididymis, seminal vesicles, and prostate did not show appreciable changes. Sperm concentration showed a gradual decline, reached severe oligospermia (fewer than 20 million/mL) after 75 d treatment, and attained uniform azoospermia after 120 d treatment. Sperm motility and viability were severely affected after 45 d treatment and reached less than 1% after 75 d treatment. The morphology of the spermatozoa by scanning electron microscopy revealed membrane damage in the acrosome, bent midpiece, coiled tail, and detached head and tail. The levels of fructose, glycerylphosphorylcholine, acid phosphatase, and lactate dehydrogenase in the seminal plasma were unaltered. Histology of the testis revealed arrest of spermatogenesis beyond the level of spermatocytes. No toxicity was evident from the haematology and serum biochemistry parameters. The libido of the treated animals was unaffected and the fertility rate was zero. The effects were comparable in both the dose regimens (Groups II and III) and were restored to normal 45 d after withdrawal of the treatment.

Sterility due to inhibition of sperm motility by oral administration of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats.

The contraceptive effects of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya have been reported in male albino rats at the dose regimens 5 and 10 mg/animal/day; oral for 150 days. The body weight, weight of testis, epididymis, seminal vesicle and ventral prostate remained unaltered during the entire course of the investigation. Total suppression of cauda epididymal sperm motility coincided with a decrease in sperm count, viability and an increase in per cent abnormal spermatozoa during 60-150 days observation period. Minor changes in the germ cell proliferations in the testis and vacuolization and pyknotic nuclei in the few epithelial cells of the cauda epididymis were observed. Histology and biochemical composition of testis and accessory sex organs, haematology and serum clinical biochemistry and serum testosterone levels remained unchanged throughout the course of the investigation. Test for estrogenicity indicated mild estrogenicity. Monthly fertility test showed negative fertility. All the altered parameters returned to normal level following 60 days withdrawal of the treatment. The results suggest that the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya exerts antifertility effects in rats without adverse toxicity and that the effects may be directly rendered on the spermatozoa.

Contraceptive evaluation and toxicological study of aqueous extract of the seeds of Carica papaya in male rabbits

The contraceptive evaluation and toxicological effects of the aqueous extract of the seeds of Carica papaya in adult male rabbits have been reported. Thirty adult male rabbits were divided into five groups of six animals each; Group I, control; Groups II-V were administered orally with aqueous extract of the seeds of C. papaya at doses of 20, 50, 75 and 100 mg/kg per day for 150 days, respectively. The body weight, reproductive organs weight, semen analysis, semen biochemistry, toxicological profiles and the fertility status have been recorded. The aqueous extract failed to exhibit contraceptive effects at any of the dose regimens tested, contrary to the observations made in the previous studies. Unaltered toxicological profiles indicated that the drug was free of side effects. The results suggest that the failure of contraceptive effects may be due to species specificity, relative resistance of the animals to the drug or lack of potency of the extract due to factors generally affect biological activity of the plant preparations.

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.

Reversible azoospermia by oral administration of the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rabbits.

Contraceptive efficacy, reversibility and toxicity, if any, of the benzene, chloroform and ethyl acetate chromatographic fractions of the chloroform extract of the seeds of Carica papaya have been investigated in adult male rabbits at a dose regimen of 50 mg/animal/day for 150 days of treatment. Body weight, semen analysis, hematology, serum clinical biochemistry and the fertility status of control and treated animals were evaluated. Chloroform and ethyl acetate chromatographic fractions did not produce appreciable changes in these parameters. However, the benzene chromatographic fraction resulted in uniform azoospermia after 15 days of treatment, which was maintained for the remainder of the 150-day observation period. The levels of fructose, glycerophosphocholine, acid phosphatase and lactate dehydrogenase in the seminal plasma were within the control range. Hematology and the serum clinical parameters showed no appreciable changes, indicating lack of toxicity. The libido of the treated animals was normal and the fertility rate was zero. Complete normalcy of altered parameters was observed 60 days following withdrawal of treatment. It is concluded that the benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya possesses reversible male contraceptive potential and the effects appear to be mediated through the testis.

Intravasal contraception with styrene maleic anhydride and its noninvasive reversal in langur monkeys (Presbytis entellus entellus)

In male langurs with azoospermia induced by vas occlusion with styrene maleic anhydride (SMA), the exploratory feasibility of azoospermia reversal by a new noninvasive reversal procedure has been assessed. Palpation, percutaneous electrical stimulation of the vas deferens, forced vibratory movement, suprapubic percussion, and per rectal digital massage of the vas deferens are the components of the multimodal noninvasive reversal procedure. The exploratory investigation reveals that a single application of the procedure leads to reversal of azoospermia. Normospermia with normal motility and viability appears after third ejaculation onwards after reversal manipulations. Ultrastructure of the spermatozoa, using scanning and transmission electron microscopy, revealed that the spermatozoa attained normalcy and sperm functional tests (i.e., hypo-osmotic swelling test, slide test for acrosome intactness, and test for sperm mitochondrial activity index) further confirmed the normalcy of the spermatozoa toward their fertilizing ability. Semen biochemistry appeared normal throughout the course of investigation. The morphology of the vas deferens, which showed exfoliation of the epithelium, was in the process of regaining normalcy after 90 days of reversal manipulations. The results suggest that noninvasive reversal technique offers the possibility of the functional azoospermia reversal within a short period of time.

Selective Killing of Leukemia and Lymphoma Cells Ectopically Expressing hCGβ by a Conjugate of Curcumin with an Antibody against hCGβ Subunit

Objective: A variety of cancers ectopically express human chorionic gonadotropin beta (hCGβ). Patients harboring such cancers have poor prognosis and adverse survival. A recombinant chimeric antibody, cPiPP, exhibiting high affinity and specificity for hCGβ/hCG was engineered. This study was designed to determine whether this antibody alone or conjugated to curcumin can selectively kill tumor cells expressing hCGβ. Experimental Design: The study was carried out on MOLT-4 and U-937 cells expressing hCGβ and on peripheral blood leukocytes of acute myeloid leukemia (AML) patients. The anticancerous compound curcumin was conjugated to cPiPP. The binding of cPiPP and cPiPP-curcumin conjugate to cells was studied by flow cytometry and cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), FACS with propidium iodide staining, trypan blue exclusion assay and microscopy. Results: The antibody did not impair the growth of MOLT-4 and U-937 cells in culture. Its conjugate with curcumin, however, was lethal to both cell lines. The immunoconjugate killed tumor cells bearing the CD33 marker of an AML patient expressing hCGβ but did not have a similar action on cells of another AML patient with the CD13 marker but who was negative for hCGβ. Conclusion: A humanized antibody against hCGβ linked to curcumin has potential for therapy of hCGβ-expressing tumors.

Ultrastructural Changes in the Spermatozoa of Langur Monkeys Presbytis entellus entellus After Vas Occlusion With Styrene Maleic Anhydride

Changes in the physical characteristics of semen and ultrastructure of the spermatozoa of langur monkeys after vas occlusion with styrene maleic anhydride (SMA), a polymer with pH-lowering action, are reported. Vas occlusion resulted in severe reversible hypospermia. Severe oligospermia was observed in the majority of animals (five of eight) in the first ejaculation, 30 days after vas occlusion, and in two animals in the second ejaculation, 60 days after vas occlusion. Subsequent monthly ejaculations for 5 months revealed uniform azoospermia. The voided spermatozoa were immotile and supravital staining confirmed necrospermia. Scanning electron microscopy (SEM) revealed severe coiling of tail, rupture of acrosomal envelope, and bent midpiece associated with damaged mitochondrial sheath. Observations by transmission electron microscopy (TEM) revealed vacuolization in the nucleus, membrane damage in the acrosome, loss of segmented columns, and numeric aberrations in the centriole of the neck, as well as degeneration of mitochondrial sheath and axoneme in the midpiece, and absence of outer plasma membrane in the midpiece and tail. The results indicate that the necrospermic status of the spermatozoa during initial ejaculations may offer instant sterility after vas occlusion with SMA.

Effects of cyproterone acetate with combination of testosterone enanthate on seminal characteristics, androgenicity and clinical chemistry in langur monkey

Daily oral administration of 1 mg/kg b.w. of cyproterone acetate and simultaneously administered testosterone enanthate (2 mg/kg b.w./15 days; i.m.) to adult male langur monkeys over a period of 90 days caused a gradual decrease in the count (to azoospermia) and motility of spermatozoa, concurrently with an increase in the percentage of non-motile as well as abnormal and immature sperm. Semen weight, volume, seminal fluid volume and circulating testosterone levels decreased nonsignificantly. Semen pH, libido and body weight remained unimpaired. The levels of SGOT, SGPT, serum alkaline phosphatase, LDH, bilirubin, Na+, K+ and hematological values did not alter significantly. All the changes were reversible. The results indicate that the combination regimen seems to affect the fertility in two ways, i.e. by inhibiting spermatogenesis in the testis and maturation process in the epididymis without altering the androgenicity.