The Interactive Effects Of Estradiol-17β Metabolites And Ascorbic Acid On Human Ovarian Cancer Cells

Ovarian cancer is one of the three most common gynecology tumors, the mortality of which is the highest. Although there have been great advances in surgical technology and therapy regimens over the last three decades, ovarian cancer remains to be the most lethal gynecological malignancy, which is largely due to the fact that the majority (-70%) of ovarian cancers are diagnosed at an advanced, chemo resistant stage. In addition, another major challenge of the current cancer treatment is that ovarian cancer cells are highly heterogeneous with distinctive biology features at the clinical, cellular and molecular levels. Thus, understanding individual subtype of ovarian cancer at these levels is critical for developing an efficacious cancer therapy based on individual type of ovarian cancer, which share similar biology features and behaviors. PART I Expression and roles of Estradiol-17p and its Metabolites in the ovarian cancerBackgroundTo date, the etiology of human ovarian cancer is poorly understood. However, estrogen is considered as a major risk factor for ovarian cancer, particularly since recent epidemiological studies have demonstrated an elevation of ovarian cancer incidence with postmenopausal usage of estrogen. Nonetheless, the exact role of estrogen in ovarian cancer is still controversial, as estrogen has been shown to have stimulatory, inhibitory, or no effect on ovarian cancer cell growth, possibly depending on individual subtype of ovarian cancer cells, doses of estrogen used, and patients’ ages (e.g., pre-vs. post-menopause).Moreover, although it is known that the majority of ovarian cancer cells express both estrogen receptor1(ESR1, also termed as ERa) and2(EiSR2, also termed as ERP), these receptors may exert opposite actions on ovarian cancer since ERa promotes ovarian cancer progression, whereas ERfi inhibits ERa-mediated stimulatory actions in ovarian cancer cells. On the other hand, recent evidence has also indicated that estrogen-induced growth of ovarian cancer cells could be independent of ER.To further complicate the situation, estrogen can be converted into many biologically active metabolites by enzymatic reactions in vivo. Specially, as catalyzed by cytochrome P450, family1, subfamily A. polypeptide1(CYP1A1) and subfamily B, polypeptide1(CYP1B1), estradiol-17pβ(E2β) can be metabolized to form the catecholestradiols2-hydroxyestradiol (2OHE2) and4-hydroxyestradiol (4OHE2), which can be subsequently converted into the methoxyestradiols2-methoxestradiol (2ME2) and4-methoxyestradiol (4ME2) by catechol-O methyltransferase (COMT). These E2β metabolites generated by cancer cells or non-cancer cells in ovary may have either pro-or anti-ovarian cancer activities as reported in many other types of cancers. Indeed,2OHE2and4OHE2at physiological concentrations stimulate ovarian cancer (OVCAR-3) cell proliferation in vitro, while2ME2at supra-physiological concentrations inhibits cell proliferation of many ovarian cancer cell lines including OVCAR-3. A2780, and primary ovarian cancer cell lines.To date, little is known about actions and roles of E2β and its metabolites in human ovarian cancer cells. Herein, we examined potential participation of E2β and its metabolites in regulating migration and proliferation of ovarian cancer cells and whether E2P and its metabolites modulate ovarian cancer cell proliferation and/or migration via ERa and ERβ.Objective:1. To examine expression of CYP1A1, CYP1B1, COMT, and estrogen receptor ERa and ERβ in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, OVCAR-429and OVCAR-432).2. To examine potential participation of E2β and its metabolites in regulating proliferation of human ovarian surface epithelial cell and ovarian cancer cells and whether E2β and its metabolites modulate ovarian cancer cell proliferation and/or migration via ERa and/or ERβ.3. To examine effects of E2P and its metabolites in regulating migration of human ovarian surface epithelial cell and ovarian cancer cells.Methods:1. Western blot analysis for expression of CYP1A1, CYP1B1, COMT, and estrogen receptor ERα and ERP in human ovarian surface epithelial (IOSE-385) and cancer cell lines (OVCAR-3, SKOV-3, OVCAR-429and OVCAR-432).2. Human ovarian surface epithelial cell and ovarian cancer cells were treated with E2P and its metabolites for6days. Cell proliferation detected using crystal violet method and then confirm whether the action via ERa and/or ERp.3. Wound healing assay was performed to confirm E2β and its metabolites’action on ovarian surface epithelial cell and ovarian cancer cells migration.Results:1. Western blotting revealed the presence of CYP1A1, CYP1B1, COMT, ERa, and ERβ in all cell lines tested.1) The levels of CYP1Al and CYP1B1were similar among all cell lines tested while CYP1B1levels were at least2fold higher than the CYP1A1levels in IOSE-385, OVCAR-3, and OVCAR-432cells.2)The level of COMT in IOSE-385cells were at least2fold greater than those in other cell lines.3)The levels of ERa and ERβ in OVCAR-432cells were higher than those in other cell lines.2.E2β and its four metabolites stimulated IOSE-385and OVCAR-3cell proliferation at all doses studied, whereas did not affect SKOV-3and OVCAR-432cell proliferation.1)E2β at all doses studied similarly stimulated IOSE-385and OVCAR-3cell proliferation with maximum responses observed at0.1nM.2)2OHE2,4OHE2, and4ME2at all doses studied also similarly promoted IOSE-385cell proliferation; however,2ME2did so only at0.1nM.3)2ME2,4OHE2, and4ME2at all doses studied also promoted OVCAR-3cell proliferation; however,2OHE2did so only at0.1and100nM.ICI significantly inhibited cell proliferative response to E2β, but not to2-OHE2,4-OHF2,2-ME2, and4-ME2. ERa blockade with1uM of MPP significantly inhibited E2β-stimulated cell proliferation in OVCAR-3, but not IOSE-385cells. In contrast. ERP blockade with1uM of PHTPP robustly attenuated E2β-stimulated cell proliferation in IOSE-385, but not OVCAR-3cells. However, neither MPP nor PHTPP affected IOSE-385and OVCAR-3cell proliferative responses to the four E2β metabolites.3.Treatments of E2β and its four metabolites up to40h did not significantly alter cell migration in all cell lines tested.Conclusions:1.E2βi and its metabolites stimulated cell proliferation but had no effect on cell migration, which indicated E2β and its metabolites may play important selectively role in the ovarian cancer;2.E2β and its metabolites modulate ovarian cancer cell proliferation via ERa and ERp. PART Ⅱ Role of Ascorbic Acid and the interactive effects with Estradiol-17p and its Metabolites in Human Ovarian CancerBackgroundVitamin C as an essential nutrient and antioxidant in human and certain animals has long been implicated in preventing and treating human cancers and ascorbic acid (AA) is the reduced form of vitamin C and it is the more predominant chemical structure in the human body. However, no conclusive clinic results were obtained so far, which is thought to be largely attributed to different levels of AA to which cancer cells are exposed. For instance, acute treatments with AA within its physiological range (<100μM) for1or2h do not have any significant impact on growth of many cancer cells including ovarian cancer cells. Conversely, AA at pharmacologic concentrations can specifically target cancer cells, but not normal cells, killing ovarian, breast, and lung cancer cells in vitro (with EC50<10mM for the majority of tumor cells tested) and suppressing cancer progression of ovarian, pancreatic, and glioblastoma cancer in mice, possibly via delivering relatively high levels of hydrogen peroxide to the cancer tissues. Thus, these data indicate that AA’s anti-cancer property is highly dependent on concentrations. Herein, our current study extend these reports, examined role of AA in regulating growth of ovarian surface epithelial cell and ovarian cancer cells.Cstradiol-17β (E2β) and ascorbic acid (AA) have been implicated in the positive and negative progression of cancer growth, respectively. To date, little is known about the interactions between E2β3and its metabolites with A A, particularly within their physiological ranges. Herein, we determined if E2β and its metabolites and AA interactively modulate ovarian cancer cell proliferation.Objective:1. To examine potential participation of AA in regulating proliferation of human ovarian surface epithelial cell and ovarian cancer cells;2. To determine if E2P and its metabolites and AA interactively modulate human ovarian surface epithelial cell and ovarian cancer cells proliferation.Methods:1.Human ovarian surface epithelial cell and ovarian cancer cells were treated with AA for4days. Cell proliferation was evaluated using the crystal violet method.2.After determination of an optimal dose, human ovarian surface epithelial cell and ovarian cancer cells were treated with E2β and its metabolites and AA for4days. Using the crystal violet method to evaluate interactive effects of E2β and its metabolites with AA.Results:1.AA stimulated apoptosis of human ovarian surface epithelial cell and ovarian cancer cells and the effects were more significant with the concentration increasing.1)As compared to the control, treatments of AA for4days suppressed (P≤0.05) cell proliferation in all doses and in all cell lines tested. The estimated IC50were60,60,67, and122μM, respectively for IOSE-385, OVCAR-3, SKOV-3, and OVCAR-432cells.2)AA can enhanced OVCAR429cells proliferation (≤125umol/L) whereas stimulated apoptosis at higher concentration (>125umol/L).2.After determination of an optimal dose, ovarian cell lines were treated with80μM of AA in the presence of0.1nM E2β and its metabolites for4days. We observed that E2β and its four metabolites significantly (P≤0.05) attenuated the AA-induced inhibition on cell proliferation in IOSK-385. OVCAR-3, and OVCAR-432cells, but not SKOV-3and OVCAR-429cells.Conclusions:1.AA stimulated apoptosis of human ovarian surface epithelial cell and ovarian cancer cells and the effects were more significant with the concentration increasing, which indicated AA may play important role in the ovarian cancer;2. E2β and its metabolites antagonize AA-suppressed cell proliferation in human ovarian surface epithelial cell and ovarian cancer cells and block the actions of E2β and its metabolites may considerably promote AA’s anti-ovarian cancer activity.