Hepatic triiodothyronine sulfation and its regulation by growth hormone and triiodothyronine in rats.

The regulatory mechanism of cytosolic sulfation of T3 has been studied in rat liver. Sulfation of T3 is sexually differentiated in adult rats of Sprague-Dawley (SD), Fisher 344, and ACI strains. In SD strain, the male animals showed 4 times higher sulfating activity than did the females. The specific activity was decreased by hypophysectomy of male adult rats, but was not affected in the females. Thus, the sex-difference was abolished in the hypophysectomized condition. Supplement of human GH intermittently twice daily for 7 days, to mimic the male secretory pattern, increased T3 sulfating activity in both sexes of hypophysectomized rats, whereas continuous infusion to mimic a female secretory pattern had no appreciable effect. Cytosolic sulfation of T3 was decreased by 25 to 30% by thyroidectomy or propylthiouracil treatment of male adult rats, and was restored by the supplementation of T3 (50 micrograms/kg daily for 7 days) to thyroidectomized rats. Administration of T3 in hypophysectomized rats almost completely restored the sulfating activity in the males and increased the activity in the females. Cytosolic T3 sulfation was inhibited by the addition of known inhibitors of phenol sulfotransferase, pentachlorophenol or 2,6-dichloro-4-nitrophenol. These results indicate a role of pituitary GH in hepatic sulfation of thyroid hormones in rats. The data obtained also raise the possibility that GH may modify the effect of thyroid hormones on the pituitary by a feed-back mechanism through changing the level of a sex-dominant phenol sulfotransferase(s) in rat livers. T3 was also sulfated in hepatic cytosols of mouse, hamster, rabbit, dog, monkey, and human.(ABSTRACT TRUNCATED AT 250 WORDS)     

Species-specific pharmacology of antiestrogens: role of metabolism

The nonsteroidal antiestrogen tamoxifen exhibits a paradoxical species-specific pharmacology. The drug is a full estrogen in the mouse, a partial estrogen/antiestrogen in humans and the rat, and an antiestrogen in the chick oviduct. Inasmuch as tamoxifen has antiestrogenic effects in vitro, differential metabolism of tamoxifen to estrogens might occur in the species in which it has an estrogenic pharmacology. Tamoxifen or its metabolite 4-hydroxytamoxifen could lose the alkylaminoethane side chain to form the estrogenic compound metabolite E or bisphenol. Sensitive metabolic studies with [3H]tamoxifen in chicks, rats, and mice identified 4-hydroxytamoxifen as the major metabolite, but no potentially estrogenic metabolites were observed. Athymic mice with transplanted human breast tumors can be used to study the ability of tamoxifen to stimulate target tissue or tumor growth. Estradiol caused the growth of transplanted MCF-7 breast cancer cells into solid tumors and a uterotrophic response. However, tamoxifen does not support tumor growth when administered alone, although it stimulates uterine growth. Since a similar profile of metabolites is sequestered in human and mouse tissues, these studies strongly support the concept that the drug can selectively stimulate or inhibit events in the target tissues of different species without metabolic intervention.     

Determination of tamoxifen and metabolites in serum by capillary electrophoresis using a nonaqueous buffer system

Tamoxifen (TAM), an antiestrogen, is widely used to treat hormone-dependent breast cancer in post-menopausal women. TAM may be used as a chemopreventive agent in women of child-bearing age; however, few data exist describing potential TAM-induced fetal toxicity. In support of the National Toxicology Program's characterization of reproductive and developmental effects of TAM, this work describes an analytical technique utilizing capillary electrophoresis (CE) for the detection of circulating levels of TAM, N-desmethyltamoxifen (DMT), and 4-hydroxytamoxifen (4-HT) in maternal rodent serum. Greater than 90% of ³H-labeled TAM was extractable from serum using 98:2 hexane–isoamyl alcohol. Optimum separation of TAM, DMT, and 4-HT was obtained on a 57 cm×50 μm capillary using a nonaqueous buffer system of 1:1 methanol–acetonitrile containing 50 mM ammonium acetate and 1% acetic acid. 4-Dimethylaminopyridine was used as internal standard. Temperature and voltage were optimized at 40°C and 15 kV, respectively. The limit of detection of TAM by UV detection at 214 nm was approximately 800 amol. TAM and DMT were confirmed in serum of female rats 4 h following a single oral dose of 120 mg/kg. Transplacental exposure of TAM to fetal tissue will be evaluated using this technique.     

Enzymatic sulfation of steroids III. The sulfation of corticosterone by the glucocorticoid sulfotranferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This ‘mosaic’ assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6–9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate.The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20–32% and 25–35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37–55% and 40–60% decreases of sulfation of the twoo steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases.Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferase I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due to mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

DNA adducts formed from 4-hydroxytamoxifen are more mutagenic than those formed by alpha-acetoxytamoxifen in a shuttle vector target gene replicated in human Ad293 cells

The drug tamoxifen, used to treat breast cancer, causes liver cancer in rats and endometrial cancer in women. Tamoxifen forms liver DNA adducts in both short- and long-term dosing of rodents, and DNA adducts have also been reported in tissues of women undergoing tamoxifen therapy. It is not known if the induction of endometrial cancer in women is through these DNA adducts or through the estrogenic nature of the drug. In this study, we have investigated the mutagenicity of two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM). These form the same DNA adducts as those found in tamoxifen-treated rats. The two compounds were used to treat the pSP189 plasmid containing the supF gene, which was replicated in Ad293 cells before being screened in indicator bacteria. Plasmid reacted with 4-OHtamQM was more likely to be mutated (2-7-fold increase) than that reacted with alpha-acetoxytamoxifen, despite having a lower level of DNA damage (12-20-fold less), as assayed by (32)P-postlabeling. The two compounds induced statistically different mutation spectra in the supF gene. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC -->TA transversions while GC-->AT transitions were formed in 4-OHtamQM-treated plasmid. 4-OHTamQM-treated DNA induced a larger proportion of multiple mutations and large deletions compared to alpha-acetoxytamoxifen. Sites of mutational hotspots were observed for both compounds. In conclusion, the quantitatively minor DNA adduct of tamoxifen (dG-N(2)-4-hydroxytamoxifen) is more mutagenic than the major tamoxifen DNA adduct (dG-N(2)-tamoxifen).     

Enzymatic sulfation of steroids. II. The sulfation of corticosterone by the glucocorticoid sulfotransferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This "mosaic" assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6--9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate. The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20--32% and 25--35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37--55% and 40--60% decreases of sulfation of the two steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases. Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferases I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

Bioactivation of 7-hydroxymethyl-12- methyibenz[a]anthracene by rat liver bile acid sulfotransferase I

The bioactivation of 7-hydroxy-methyl-12-methylbenz[a]anthracene (HMBA) to an electrophilic sulfuric acid ester metabolite has been shown to be catalyzed by rat liver bile acid sulfotransferase I (BAST I). The sulfation and activation of HMBA by BAST I was determined by the ability of sulfated HMBA to form DNA ad-ducts. The BAST I was also shown to react with rabbit anti-human dehydroepiandrosterone sulfotransferase antisera and to represent a major form of hydroxysteroid/bile acid sulfotransferase in female rat liver cytosol. Higher levels of BAST I activity and immunoreactivity as well as HMBA-DNA adduct formation were detected in female rat liver cytosol than in male rat liver cytosol. The bioactivation of HMBA by pure BAST I was dependent on the presence of 3′-phosphoadenosine 5′-phos-phosulfate (PAPS) in the reaction and was inhibited by dehydroepiandrosterone, a physiological substrate for BAST I. Glutathione, a cellular nucleophile with important protective properties, decreased DNA adduct formation in the HMBA sulfation reaction in the absence of glutathione S-transferase activity. These results indicate the usefulness of BAST I to investigate the sulfation and activation of HMBA and probably other hydroxy-methylated polyaromatic hydrocarbons to electrophilic and mutagenic metabolites under defined reaction conditions.     

Sulfation of tibolone and tibolone metabolites by expressed human cytosolic sulfotransferases

Tibolone is an important therapeutic agent used in the treatment of menopausal symptoms in many countries and has beneficial effects on menopausal and postmenopausal vasomotor, bone, vaginal and mood symptoms without affecting the endometrial, breast or cardiovascular systems. The rapid metabolism of tibolone to active metabolites including 3-OH-tibolone, 3β-OH-tibolone and Δ⁴-tibolone may be important in its tissue-specific effects. Sulfation also has a major role in the metabolism and regulation of the tissue-specific activity of tibolone and its metabolites. The ability of seven major expressed human sulfotransferase (SULT) isoforms to sulfate tibolone and its three metabolites was examined. Expressed human SULT2A1 was capable of sulfating tibolone and all three metabolites with the highest affinity for 3-OH-tibolone. SULT1E1 conjugated both 3-OH-tibolone metabolites and tibolone itself slightly. SULT2B1b sulfated both 3-OH metabolites but not tibolone or Δ⁴-tibolone. SULT isoforms 1A1, 1A3, 1B1 and 1C1 did not demonstrate detectable activity. Sulfation of tibolone and its metabolites by human tissue cytosols was analyzed to determine whether the pattern of tibolone sulfation corresponded to the known expression of SULT isoforms in each tissue. The tissue-specific effects of tibolone may be regulated in part by the inactivation of tibolone and its metabolites by specific human SULT isoforms.     

In vivo and in vitro oxidative regulation of rat aryl sulfotransferase IV (AST IV)

Sulfotransferase catalyzed sulfation is important in the regulation of different hormones and the metabolism of hydroxyl containing xenobiotics. In the present investigation, we examined the effects of hyperoxia on aryl sulfotransferase IV in rat lungs in vivo. The enzyme activity of aryl sulfotransferase IV increased 3- to 8-fold in >95% O2 treated rat lungs. However, hyperoxic exposure did not change the mRNA and protein levels of aryl sulfotransferase IV in lungs as revealed by Western blot and RT-PCR. This suggests that oxidative regulation occurs at the level of protein modification. The increase of nonprotein soluble thiol and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios in treated lung cytosols correlated well with the aryl sulfotransferase IV activity increase. In vitro, rat liver cytosol 2-naphthol sulfation activity was activated by GSH and inactivated by GSSG. Our results suggest that Cys residue chemical modification is responsible for the in vivo and in vitro oxidative regulation. The molecular modeling structure of aryl sulfotransferase IV supports this conclusion. Our gel filtration chromatography results demonstrated that neither GSH nor GSSG treatment changed the existing aryl sulfotransferase IV dimer status in cytosol, suggesting that oxidative regulation of aryl sulfotransferase IV is not caused by dimer-monomer status change.     

Estrone sulfatase versus estrone sulfotransferase in human breast cancer: potential clinical applications

Estrone sulfate (E₁S) is concentrated in high levels in human breast cancer tissue. The values are particularly high in postmenopausal women and many times those circulating in the plasma. Also, the tissular concentration of this conjugate are significantly higher in tumoural tissue than in the area of the breast considered as normal. The enzyme which hydrolyzes E₁S: sulfatase, as well as the enzyme which biosynthesises this conjugate: sulfotransferase, are present in significant concentrations in breast cancer tissue. Consequently, E₁S is a balance between the activities of the two enzymes. As breast cancer tissue has all the enzymes necessary for the synthesis of estradiol (E₂), and the formation of E₂ from E₁S ‘via sulfatase’ is the main pathway, it was very attractive to explore inhibitory agents of this enzyme. It was observed that different substances including antiestrogens (4-hydroxytamoxifen, ICI 164,384) and various progestins (promegestone, nomegestrol acetate, medrogestone) as well as Org OD14 (tibolone) can block the sulfatase activity. In addition, it was demonstrated that different progestins (medrogestone, nomegestrol acetate, TX-525) and org OD14 can stimulate the sulfotransferase activity for the formation of the biologically inactive E₁S. It is concluded that the inhibition of sulfatase and the stimulation of sulfotransferase activity can open interesting possibilities to explore these effects in patients with breast cancer.     

In vitro and in vivo binding of toremifene and its metabolites in rat uterus

The in vitro binding affinities of toremifene (TOR), 4-hydroxy toremifene (4-OH-TOR) and several other metabolites for the rat uterine cytosolic estrogen receptor were compared with those of tamoxifen (TAM) and 4-hydroxy tamoxifen (4-OH-TAM). Only small differences were observed and the binding affinities of both 4-hydroxy metabolites were similar to that of estradiol (E2). Uterine uptake and subcellular distribution of [3H]TOR and [3H]TAM were then compared at 1, 8 and 72 h after administration to castrated rats. The uptake and retention of both antiestrogens were similar at all times. In each case the amount of nuclear bound radioactivity declined to low levels at 8 and 72 h but the ratios of 4-OH-TAM/TAM and 4-OH-TOR/TOR determined by HPLC analysis increased dramatically at 72 h. The level of radioactivity in both plasma and uterine cytosol at 72 h was significantly higher following [3H]TAM administration. However, most of the radioactivity appeared to be in a conjugated form since it was not extractable with solvent. Finally, the ability of prior administration of each antiestrogen (100 mg/kg) to block uterine [3H]estradiol uptake was examined at 3 and 7 days. It was found that uterine wet weights were higher than control one week after administration of both compounds. Prior administration of TOR increased nuclear uptake of [3H]E2 whereas TAM had no effect. The results of these experiments suggest that toremifene and tamoxifen have very similar in vitro and in vivo binding properties but differences in metabolism exist that may be important.     

KLF4 overcomes tamoxifen resistance by suppressing MAPK signaling pathway and predicts good prognosis in breast cancer

Tamoxifen resistance represents a daunting challenge to the successful treatment for breast cancer. Krüppel-like factor 4 has critical roles in the development and progression of breast cancer, but its expression, function and regulation in the efficacy of TAM therapy in breast cancer have yet to be investigated. Here, we examined the clinical significance and biologic effects of KLF4 in breast cancer. Firstly, higher expression of KLF4 correlated with increased TAM sensitivity in breast cancer cells, and analysis of GEO datasets indicated that KLF4 expression was positively correlated with ERα and enhanced expression of KLF4 sensitized breast cancer patients to endocrine therapy. Knockdown of KLF4 in MCF-7 and BCAP37 cells led to increased TAM resistance, while ectopic KLF4 expression promoted the responsiveness to TAM in T47D and TAM-resistant MCF-7/TAM cells. Secondly, ectopic KLF4 overexpression suppressed MCF-7/TAM cell growth, invasion and migration. Moreover, KLF4 expression was down-regulated in breast cancer tumor tissues and high expression of KLF4 was associated with favorable outcomes. Mechanistically, KLF4 may enhance the responsiveness of breast cancer cells to TAM through suppressing mitogen-activated protein kinase (MAPK) signaling pathway. We found that ERK and p38 were more activated in MCF-7/TAM compared with MCF-7, and treatment with MAPK-specific inhibitors significantly suppressed cell viability. Knockdown of KLF4 activated ERK and p38 and drove MCF-7 cells to become resistant to TAM. Conversely, overexpression of KLF4 in MCF-7/TAM cells suppressed ERK and p38 signaling and resulted in increased sensitivity to TAM. Therefore, our findings suggested that KLF4 contributed to TAM sensitivity in breast cancer via phosphorylation modification of ERK and p38 signaling. Collectively, this study highlighted the significance of KLF4/MAPK signal interaction in regulating TAM resistance of breast cancer, and suggested that targeting KLF4/MAPK signaling may be a potential therapeutic strategy for breast cancer treatment, especially for the TAM-resistant patients.     

Inhibition of human breast cancer cell proliferation with estradiol metabolites is as effective as with tamoxifen.

The anti-estrogenic substance tamoxifen is effective in the adjuvant therapy applied in human breast cancer. Since it partly exhibits estrogenic activity and has serious side-effects, however, pure anti-estrogenic compounds are being sought. In our experimental study, we compared the anti-proliferative effect of estradiol and 13 endogenous estradiol metabolites on human breast cancer cells with the effect of tamoxifen. We used MCF-7 and MDA-MB 231, the well-established estrogen receptor-positive and -negative cell lines. 4-hydroxytamoxifen, the active metabolite of tamoxifen, estradiol and 13 estradiol metabolites were tested in concentrations ranging from 3.1 to 100 microM. Incubation time was 4 days and cell proliferation was measured by means of the ATP chemosensitivity test. 4-hydroxytamoxifen showed an IC50 value of 27 microM and 18 microM in MCF-7 and MDA-MB 231 cells, respectively. Estradiol and its metabolites were anti-proliferative in both cell lines. A few A-ring metabolites were more effective in inhibiting cell proliferation than D-ring metabolites and the parent substance 17beta-estradiol. 4-OHE1, 2-MeOE1 and 2-MeOE2 were as effective in both cell lines as tamoxifen. For the first time it has been demonstrated that endogenous estradiol metabolites are equally anti-proliferative as tamoxifen in the context of human breast cancer cells. Since some of these metabolites exhibit no estrogenic activity, they are likely to be valuable in clinical studies of chemoprevention and adjuvant therapy of breast cancer.     

Enhancement of Apoptotic and Autophagic Induction by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin in Human Breast Adenocarcinoma and Neuroblastoma Cells with Tamoxifen

Breast cancer is one of the most common cancers amongst women in North America. Many current anti-cancer treatments, including ionizing radiation, induce apoptosis via DNA damage. Unfortunately, such treatments are non-selective to cancer cells and produce similar toxicity in normal cells. We have reported selective induction of apoptosis in cancer cells by the natural compound pancratistatin (PST). Recently, a novel PST analogue, a C-1 acetoxymethyl derivative of 7-deoxypancratistatin (JCTH-4), was produced by de novo synthesis and it exhibits comparable selective apoptosis inducing activity in several cancer cell lines. Recently, autophagy has been implicated in malignancies as both pro-survival and pro-death mechanisms in response to chemotherapy. Tamoxifen (TAM) has invariably demonstrated induction of pro-survival autophagy in numerous cancers. In this study, the efficacy of JCTH-4 alone and in combination with TAM to induce cell death in human breast cancer (MCF7) and neuroblastoma (SH-SY5Y) cells was evaluated. TAM alone induced autophagy, but insignificant cell death whereas JCTH-4 alone caused significant induction of apoptosis with some induction of autophagy. Interestingly, the combinatory treatment yielded a drastic increase in apoptotic and autophagic induction. We monitored time-dependent morphological changes in MCF7 cells undergoing TAM-induced autophagy, JCTH-4-induced apoptosis and autophagy, and accelerated cell death with combinatorial treatment using time-lapse microscopy. We have demonstrated these compounds to induce apoptosis/autophagy by mitochondrial targeting in these cancer cells. Importantly, these treatments did not affect the survival of noncancerous human fibroblasts. Thus, these results indicate that JCTH-4 in combination with TAM could be used as a safe and very potent anti-cancer therapy against breast cancer and neuroblastoma cells.     

Liquid chromatography-mass spectrometry method for the quantification of tamoxifen and its metabolite 4-hydroxytamoxifen in rat plasma: application to interaction study with biochanin A (an isoflavone)

Tamoxifen is the agent of choice for the treatment of estrogen receptor-positive breast cancer. Tamoxifen is a substrate of P-glycoprotein (P-gp) and microsomal cytochrome P450 (CYP) 3A, and biochanin A (BCA) is an inhibitor of P-gp and CYP3A. Hence, it could be expected that BCA would affect the pharmacokinetics of tamoxifen. In the present study we have developed and validated a simple, sensitive and specific LC-ESI-MS/MS method for the simultaneous quantification of tamoxifen and its metabolite 4-hydroxytamoxifen with 100 μL rat plasma using centchroman as an internal standard (IS). Tamoxifen, 4-hydroxytamoxifen and IS were separated on a Supelco Discovery C18 (4.6 mm × 50 mm, 5.0 μm) column under isocratic condition using 0.0 1M ammonium acetate (pH 4.5):acetonitrile (10:90, v/v) as a mobile phase. The mobile phase was delivered at a flow rate of 0.8 mL/min. The method was proved to be accurate and precise at linearity range of 0.78-200 ng/mL with a correlation coefficient (r) of ≥ 0.996. The intra- and inter-day assay precision ranged from 1.89 to 8.54% and 3.97 to 10.26%, respectively; and intra- and inter-day assay accuracy was between 87.63 and 109.06% and 96 and 103.89%, respectively for both the analytes. The method was successfully applied to study the effect of oral co-administration of BCA (an isoflavone) on the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen in female rats. The coadministration of BCA caused no significant changes in the pharmacokinetics of tamoxifen and 4-hydroxytamoxifen. However, the peak plasma concentration (C(max)) of 4-hydroxytamoxifen in BCA pretreated rats was significantly (P<0.05) lower than those from control group.     

Effects of tamoxifen and 4-hydroxytamoxifen on the pNR-1 and pNR-2 estrogen-regulated RNAs in human breast cancer cells

The estrogenic and antiestrogenic activities of tamoxifen and 4-hydroxytamoxifen have been measured on the expression of two estrogen-regulated RNAs (pNR-1 and pNR-2) in the MCF7 human breast cancer cell line cultured in phenol red-free medium. The two antiestrogens increased the level of the pNR-1 RNA to about 80% of the estradiol-induced level, and the induction by estradiol was not significantly antagonized by either antiestrogen. In contrast, the pNR-2 mRNA was only increased to about 10% of the estradiol-induced level, and its induction by estradiol was antagonized by both tamoxifen and 4-hydroxytamoxifen. Thus, the two RNAs respond in dramatically different ways to these antiestrogens. 4-Hydroxytamoxifen and estradiol have similar affinities for the estrogen receptor; however, the induction of both RNAs by 4-hydroxytamoxifen required a 10-fold higher concentration than estradiol for maximum agonist activity, and a 500-fold molar excess was required to antagonize the induction by estradiol. Tamoxifen has a 20-100-fold lower affinity than estradiol for the estrogen receptor. A 200-fold higher concentration was required for maximum agonist activity and a 10,000-fold molar excess to antagonize the induction by estradiol. These results emphasize the complexity of antiestrogen action in human breast cancer cells.     

DLG5 suppresses breast cancer stem cell‐like characteristics to restore tamoxifen sensitivity by inhibiting TAZ expression

Tamoxifen (TAM) is a primary drug for treatment of estrogen receptor positive breast cancer. However, TAM resistance remains a serious threat to breast cancer patients and may be attributed to increased stemness of breast cancer. Here, we show that discs large homolog 5 (DLG5) expression is down‐regulated in TAM‐resistant breast cancer and cells. DLG5 silencing decreased the sensitivity to TAM and increased the frequency and stemness of CD44⁺/CD24^? breast cancer stem cells (BCSCs) and TAZ, a transducer of the Hippo pathway, expression in MCF7 cells while DLG5 overexpression had opposite effects. TAZ silencing restored the sensitivity to TAM and reduced the frequency and stemness in TAM‐resistant breast cancer cells. Taken together, our data indicate that down‐regulated DLG5 expression increases the stemness of breast cancer cells by enhancing TAZ expression, contributing to TAM resistance in breast cancer.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Hepatoprotective effect of green tea (Camellia sinensis) extract against tamoxifen-induced liver injury in rats

Tamoxifen citrate (TAM), is widely used for treatment of breast cancer. It showed a degree of hepatic carcinogenesis. The purpose of this study was to elucidate the antioxidant capacity of green tea (Camellia sinensis) extract (GTE) against TAM-induced liver injury. A model of liver injury in female rats was done by intraperitoneal injection of TAM in a dose of 45mg Kg(-1) day(-1), i.p. for 7 successive days. GTE in the concentration of 1.5 %, was orally administered 4 days prior and 14 days after TAM-intoxication as a sole source of drinking water. The antioxidant flavonoid; epicatechin (a component of green tea) was not detectable in liver and blood of rats in either normal control or TAM-intoxicated group, however, TAM intoxication resulted in a significant decrease of its level in liver homogenate of tamoxifenintoxicated rats. The model of TAM-intoxication elicited significant declines in the antioxidant enzymes (glutathione-S-transferase,glutathione peroxidase, superoxide dismutase and catalase) and reduced glutathione concomitant with significant elevations in TBARS (thiobarbituric acid reactive substance) and liver transaminases; sGPT (serum glutamate pyruvate transaminase) and sGOT (serum glutamate oxaloacetate transaminase) levels. The oral administration of 1.5 % GTE to TAM-intoxicated rats, produced significant increments in the antioxidant enzymes and reduced glutathione concomitant with significant decrements in TBARS and liver transaminases levels. The data obtained from this study speculated that 1.5 % GTE has the capacity to scavenge free radical and can protect against oxidative stress induced by TAM intoxication. Supplementation of GTE could be useful in alleviating tamoxifen-induced liver injury in rats.     

Cx43 deficiency confers EMT-mediated tamoxifen resistance to breast cancer via c-Src/PI3K/Akt pathway

Tamoxifen (TAM) resistance has indicated a significant challenge during endocrine therapy for hormone-sensitive breast cancer. Thus, it is significant to elucidate the molecular events endowing TAM resistance to endocrine therapy. In this study, we found that epithelial-mesenchymal transition (EMT) was an important event to confer TAM resistance, and attenuating EMT by elevating connexin (Cx) 43 expression could reverse TAM resistance. Specifically, Cx43 overexpression improved TAM sensitivity, while Cx43 depletion facilitated TAM insensitivity by modulating EMT in T47D TAM-resistant and -sensitive cells, and transplanted xenografts. Importantly, we found a novel reciprocal regulation between Cx43 and c-Src/PI3K/Akt pathway contributing to EMT and TAM resistance in breast cancer. Moreover, we identified that Cx43 deficiency was significantly correlated with poor relapse-free survival in patients undergoing TAM treatment. Therefore, Cx43 represents a prognostic marker and an attractive target for breast cancer treatments. Therapeutic strategies designed to increase or maintain Cx43 function may be beneficial to overcome TAM resistance.