Young C. Lin

Antiproliferative activity of gossypol and gossypolone on human breast cancer cells.

Gossypol is a polyphenolic aldehyde occurring naturally in cottonseed that produces antisteroidogenic activity in vivo, has been extensively investigated as a male contraceptive agent, and has demonstrated anticancer activity. Gossypolone, the major metabolite of gossypol, also prossesses antisteroidogenic activity but has not been examined for its anticancer properties. The objectives of these investigations are to compare the effects of gossypolone with those of gossypol on cell proliferation of hormone-dependent and hormone-independent human breast carcinoma cells, i.e., MCF-7, MCF-7 Adr and MDA-MB-231 cells. Gossypol and gossypolone were examined at concentrations up to 10 microM, and cellular DNA synthesis was monitored by 3H-thymidine incorporation. Gossypol and gossypolone produced dose-dependent suppression of DNA synthesis in all of the human breast cell lines examined. Gossypol produced potent antiproliferative activity in MCF-7 cells at doses as low as 30 nM. Co-incubation of MCF-7 cells with gossypol (5 microM) and estradiol (10 nM) did not alter the effects of gossypl. Treatment of human breast cancer cells with 2.5 microM of gossypol resulted in alterations in cell shape and attachment to the surface of the culture dishes. At gossypol doses of 10 microM, pericytoplasmic globuation and cytoplasmic swelling were observed in the majority of breast cancer cells. These changes in cellular morphology indicate a loss of ability of the cells to maintain normal cell membrane permeability, resulting in subsequent disorganization and loss of cytoplasmic organelles. Gossypolone is less potent than gossypol in producing these effects in the human breast cancer cell lines, whereas it possesses equipotent antisteroidogenic and antireproductive activities with gossypol. These investigations suggest that gossypol and gossypol analogs may have therapeutic potential for human breast cancer.

Antioxidant properties of peptide fractions from silver carp (Hypophthalmichthys molitrix) processing by-product protein hydrolysates evaluated by electron spin resonance spectrometry.

Silver carp processing by-product protein is usually discarded as an industrial solid waste. In this study the protein was recovered using a pH-shift method, after which seven commercial proteases were separately employed to prepare antioxidative hydrolysates. Among the hydrolysates, pepsin hydrolysates, which had the highest free radical-scavenging activity, were further separated into five peptide fractions, SCPH-I (>10kDa), SCPH-II (5-10kDa), SCPH-III (3-5kDa), SCPH-IV (1-3kDa), and SCPH-V (<1kDa), by using ultrafiltration. The antioxidative properties of the peptide fractions were investigated, using a free radical-scavenging assay, by electron spin resonance. The results show that SCPH-V had the highest scavenging effects on DPPH (1,1-diphenyl-2-picrylhydrazyl), hydroxyl and superoxide anion radicals. SCPH-V had potent antioxidant activity in the prevention of the peroxidation of linoleic acid and alleviation of H2O2-induced oxidative stress in human intestinal epithelial caco-2 cells. The results indicated that the antioxidant capacity of silver carp by-product hydrolysates could be enhanced by ultrafiltration.

Gossypol in female fertility control: Ovum implantation and early pregnancy inhibited in rats

Intramuscular administration of gossypol to normally cycling female rats induced an irregularity of the cyclic pattern for as long as the treatment was continued. Furthermore, administration of gossypol from days 0 (day of sperm-positive vaginal smear) to 8 of pregnancy prevented the normal maintenance of pregnancy. Serum values of progesterone and estradiol 17 beta in gossypol-treated normally cycling and pregnant rats were significantly lower than the control levels. The supplement of a combination of exogenous progesterone and estradiol 17 beta eliminated the inhibitory effects of gossypol on ovum implantation and the maintenance of pregnancy. Our results indicate that gossypol may have some usefulness in female fertility control.

Transformation of MCF-10A human breast epithelial cells by zeranol and estradiol-17beta.

Abstract:  Among the endocrine factors associated with breast cancer, estrogens are considered to play a central role in human breast carcinogenesis. Breast cancer risks are increased by long‐term exposure to estrogens. Zeranol (Ralgro) is a nonsteroidal agent with estrogenic activity that is used as a growth promoter in the U.S. beef and veal industry. To determine whether zeranol and estradiol‐17β play a role in the neoplastic transformation of human breast and to compare the estrogenic potency of zeranol to that of estradiol‐17β in human breast, we treated human breast epithelial cell MCF‐10A with different doses of zeranol or estradiol‐17β for 10 repeated treatment cycles. By utilizing the doubling time assay, soft agar assay, and reverse transcriptase polymerase chain reaction (RT‐PCR) assay, we showed that 10 repeated estradiol‐17β or zeranol treatment cycles to MCF‐10A cells decrease the doubling time of the cells by 30 to 40% and stimulate colony formation in soft agar and induce estrogen receptor β (ER‐β) mRNA expression, all of which are not dose related in our tested dose range. Furthermore, we show that zeranol and estradiol‐17β have a similar potency in the stimulation and inhibition of gene expressions in human breast cancer cell line MCF‐7 by RT‐PCR. These results indicate that both zeranol and estradiol‐17β can induce human breast epithelial cell neoplastic transformation with similar potency in the long‐term exposure through the oxidation‐reduction (redox) pathway and/or ER‐β‐mediated pathway. 

Translational studies on aromatase, cyclooxygenases, and enzyme inhibitors in breast cancer☆

Aromatase expression and enzyme activity in breast cancer patients is greater in or near the tumor tissue compared with the normal breast tissue. Regulation of aromatase expression in human tissues is quite complex, involving alternative promoter sites that provide tissue-specific control. Previous studies in our laboratories suggested a strong association between aromatase (CYP19) gene expression and the expression of cyclooxygenase (COX) genes. Our hypothesis is that higher levels of COX expression result in higher levels of prostaglandin E2 (PGE2), which in turn increases CYP19 expression through increases in intracellular cyclic AMP levels. This biochemical mechanism may explain the beneficial effects of non-steroidal anti-inflammatory drugs (NSAIDs) on reducing the risks of breast cancer. The effects of NSAIDs (ibuprofen, piroxicam, and indomethacin), a COX-1 selective inhibitor (SC-560), and COX-2 selective inhibitors (celecoxib, niflumic acid, nimesulide, NS-398, and SC-58125) on aromatase activity and CYP19 expression were investigated in breast cancer cell culture systems. Dose-dependent decreases in aromatase activity were observed following treatment with an NSAID or COX inhibitor, with the most effective agents being COX selective inhibitors. Real time PCR analysis of aromatase gene expression showed a significant decrease in mRNA levels in treated cells when compared to vehicle control. These results suggest that the effect of COX inhibitors on aromatase occurs at the transcriptional level. To further probe these interactions, short interfering RNAs (siRNA) were designed against either human CYP19 mRNA or human COX-2 mRNA. Treatment of breast cancer cells with aromatase siRNAs suppressed CYP19 mRNA and aromatase enzyme activity. Finally, treatment with COX-2 siRNAs downregulated the expression of COX-2 mRNA; furthermore, the siCOX-2-mediated suppression of COX-2 also resulted in suppression of aromatase mRNA. In summary, pharmacological regulation of aromatase and cyclooxygenases can act locally in an autocrine fashion to decrease the biosynthesis of estrogen and may provide additional therapy options for patients with hormone-dependent breast cancer.

Inhibitory effect of gossypol on steroidogenic pathways in cultured bovine luteal cells

Gossypol inhibits the reproductive system and steroidogenesis in both sexes. The present study investigated some possible sites subsequent to cAMP formation at which gossypol may inhibit progesterone biosynthesis. Bovine luteal cells were cultured with dibutyryl cAMP (dbcAMP), 25-OH cholesterol, or pregnenolone in the presence or absence of gossypol. Gossypol, at 17-34 microM, inhibited dbcAMP-induced progesterone secretion. Gossypol significantly inhibited the conversions of exogenous 25-OH cholesterol and pregnenolone to progesterone. However, the conversion of 25-OH cholesterol to pregnenolone was not significantly inhibited by gossypol at low doses (less than or equal to 34 microM). These results suggest that gossypol inhibits progesterone synthesis in bovine luteal cells by suppressing steroidogenic enzyme activity.

Selective vagal postganglionic innervation of the sinoatrial and atrioventricular nodes in the non-human primate

The distribution of parasympathetic postganglionic nerves to the atrioventricular (AVN) and sinoatrial nodal (SAN) regions was investigated in the non-human primate heart. Eight male monkeys (Macaca fascicularis) weighing 5.5-7.0 kg. were anesthetized (alpha-chloralose, 50 mg/kg and urethane, 500 mg/kg) and instrumented to measure arterial pressure, electrocardiogram, atrial and ventricular electrograms. The cervical vagi were electrically stimulated (20 Hz, 4 V, 2 ms) before and after selective denervation (D) of the AVN and/or SAN. Vagal stimulation was repeated during atrial pacing to assess parasympathetic modulation of AVN conduction. Ablation of parasympathetic pathways to the AVN, accomplished by the disruption of the epicardial fat and surface muscle layer at the junction of the inferior vena cava and inferior left atrium eliminated (P less than 0.01) the dromotropic effects of vagal stimulation without affecting the heart rate response (right vagus, before D, paced: atrial rate 218.0 +/- 6.3, ventricular rate 67.1 +/- 23.7; after D: atrial rate 210.3 +/- 6.4, ventricular rate 210.3 +/- 6.4 beats/min, means +/- S.D.). In sharp contrast, surgical dissection of the fat pad overlying the right pulmonary vein-superior vena cava junction significantly (P greater than 0.01) attenuated negative chronotropic effects of vagal stimulation (left vagus, before D the R-R interval increased by 832.7 +/- 146.4 ms, 209.5% increase; after D 37.4 +/- 18.0 ms, 8.8% increase). These data demonstrate discrete vagal efferent pathways innervate both the SAN and AVN regions of the non-human primate heart.

Effects of larval tapeworm (Taenia taeniaeformis) infection on reproductive functions in male and female host rats.

This report examined the effects of larval tapeworm infection on the reproductive functions in both male and female host rats. Female rats were matched by age, then randomly assigned to control and treatment groups (infected with larval tapeworms). Estrous cycles were determined by vaginal smear with 95% of the control group exhibiting 4-day normal cyclicity and only 55% of the treated group exhibiting normal cycles. Female fertility was then evaluated for the normally cycling rats based on the percentage of successful matings on the evening of proestrus, number of implantation sites on Day 8 of pregnancy, and number of pups born at term. The normally cycling rats exhibited 96% successful mating, 12.95 +/- 1.80 implantation sites, and 11.20 +/- 1.80 pups born. Five months after larval tapeworm infection, the fertility parameters were decreased to 79%, 9.10 +/- 1.20, and 7.50 +/- 1.50, respectively. The control females were then used in a study of male fertility after larval tapeworm infection employing the same parameters used to test female fertility. At the onset of the study, control groups exhibited 95% successful mating, 12.50 +/- 1.50 implantation sites, and 11.60 +/- 1.60 pups born at full term. After the 5-month infection period, the parameters were substantially reduced to 29%, 6.20 +/- 0.80 implantation sites, and 5.10 +/- 0.80 pups, respectively. Average testosterone concentrations in serum and testis from control male rats were 8.80 +/- 0.95 ng/ml and 3.88 +/- 0.25 ng/mg protein, respectively. After the 5-month infection period, these levels were reduced to 2.47 +/- 0.31 ng/ml and 1.28 +/- 0.12 ng/mg protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Fertilizability and subsequent developmental ability of bovine oocytes matured in medium containing epidermal growth factor (EGF).

We have previously shown that epidermal growth factor (EGF) is capable of promoting maturation of bovine cumulus-oocyte complexes in chemically defined serum-free medium. In this study, fertilizability and subsequent developmental capacity of bovine oocytes matured in EGF-containing medium were evaluated. Fetal bovine serum (FBS, 10%) and EGF at 10 ng/ml in Dulbeccos modified Eagles medium with Hams nutrient mixture F-12 (DME/F12) significantly increased the rate of formation of two pronuclei compared with the rate obtained from DME-F12 alone (P<0.05). Early embryonic development was assessed during 48 h in culture. Data were evaluated in terms of cleavage and four- to eight-cell formation. Oocytes matured in 10 ng/ml EGF showed significantly higher rates of cleavage (P<0.01) and four- to eight-cell formation than did oocytes matured in control medium (P<0.05). Bovine oocytes matured in the presence of EGF can be normally fertilized and can cleave and develop in vitro up to the eight-cell stage.

Effect of epidermal growth factor (EGF) and defined simple media on in vitro bovine oocyte maturation and early embryonic development

The purpose of this study is to evaluate the effect of EGF and defined simple media on in vitro bovine oocyte maturation and early embryonic development. Bovine follicular oocytes were matured in vitro and co-cultured with frozen-thawed bull sperm, which was capacitated with Hepes buffered saline (HBS) solution. After incubation of oocyte-sperm complexes for 4 days, the cleavage rate was evaluated. The results obtained were as follows: 1) When bovine oocytes were matured and embryos were developed in Park-Lin medium 1 (PL(1)) containing fetal calf serum (FCS) or EGF + bovine serum albumin (BSA), the latter treatment was more effective in inducing embryonic cleavage (18%) than FCS alone (10%). 2) When bovine oocytes were matured in Park-Lin medium 2 (PL(2)) without EGF and the subsequent embryos were developed in PL(2) medium with EGF, the cleavage rate was 22.6%. 3) When bovine oocytes were matured in PL(2) medium with EGF and then the embryos were developed in PL(2) medium with EGF, the cleavage rate was 35.8%. 4) When bovine oocytes were matured in Park-Lin medium 3 (PL(3)) without EGF and then the embryos were developed in PL(3) medium, the cleavage rate was 50%. 5) When bovine oocytes and resulting embryos were matured in PL(3) medium with EGF, the cleavage rate was 53%. 6) The parthenogenesis rate induced by PL(3) medium in our current study was comparable to the findings reported by other laboratories. These results suggest that EGF stimulates in vitro bovine oocyte maturation and subsequently affects embryonic development. It is suggested that PL(3) medium is a better defined simple medium than the other media currently used by other laboratories for in vitro bovine oocyte maturation.