Durante mucho tiempo no había principios uniformes para la Atribución de nombres a los antibióticos https://antibioticos-wiki.es . Más a menudo se les llama por el nombre genérico o especie del producto, con menos frecuencia-de acuerdo con la estructura química. Algunos antibióticos se nombran de acuerdo con el lugar donde se asignó el producto.
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PXR Influences Endocrine Therapies in Breast Cancer
PXR Influences The Efficiency of Endocrine
Karin Höckenström
Department of medical nutrition, Karolinska Institute.
The summer science school with biomedical orientation at KI, 2000.
Supervisor: Dr Jeanette Webster, department of medical nutrition, Karolinska Institute, 141 86 Huddinge. Tel: 08-585 837 18, 08-585 837 12 PXR Influences Endocrine Therapies in Breast Cancer The Human Orphan Receptor PXR Influences The Efficiency of Endocrine Therapies in Breast Cancer Karin Höckenström Department of Medical nutrition, Karolinska Institute, 141 86 Huddinge. Abstract PXR (Pregnane X receptor) is known to induce expression of the drug metabolizing enzyme CYP3A (Cytochrome P450 monooxygenase 3A). PXR mRNA has been detected in both normal and neoplastic human breast tissue. CYP3A4 enzymes have also been found in human breast cancer tissue. It was suggested that there might be a PXR/CYP3A regulated pathway that would influence the efficiency of drug therapy in breast cancer (Dotzlaw et al. 1999). The data presented here shows that tamoxifen and its active metabolite 4-hydroxytamoxifen can activate PXR at a micromolar range similar to rifampicin that is a known activator of PXR. Additional data shows that PXR is present in BK MCF-1 breast cancer cell line and that it is active. Tamoxifen is an anti-oestrogen and the major drug used in treatment for breast cancer. It is estimated that more than seven million patients undergo tamoxifen treatment every year (Gradishar and Jordan, 1997). Two significant side effects with tamoxifen are uterine cancer and tamoxifen resistance. A majority of the patients that are treated with tamoxifen under a longer period eventually become resistant. As yet the mechanism that causes tamoxifen resistance is unknown. Tamoxifen is a partial agonist and has been shown to induce expression of the multidrug resistance protein MRP2 in monkey liver. It is possible that PXR is responsible for the agonist properties of tamoxifen and that it could be involved in tamoxifen resistance. Introduction
When the ligand is in place, the receptor
protein undergoes a structural change and
progesterone and cortisol regulate a number
of processes in higher eukaryotes including
homeostasis and differentiation. They are
where it binds to a short DNA sequence called
Hormone Response Element (HRE) and there
receptor protein in the cytoplasm. A typical
it regulates the transcription of target genes.
target cell contains 10,000 steroid receptors.
PXR Influences Endocrine Therapies in Breast Cancer Fig. 2 The two isoforms of human PXR. The numbers represent the amino acids residues. DBD, DNA binding domain. PXR 2 has a deletion of 123 nucleotides in the LBD, ligand Fig. 1 The steroid hormone receptor can be located either in the cytoplasm or in the
PXR is most abundant in liver and intestine,
(Bertilsson et al. 1998) yet PXR mRNA has
from human liver cDNA library. PXR is a novel
neoplastic breast tissue (Dotzlaw et al.
orphan nuclear receptor since it lacks an
1999). Nuclear receptors regulate metabolic
pathways by regulating the transcription of
activated in vitro by both naturally occurring
progesterone and synthetic glucocorticoids at
transcription of the CYP3A genes that encode
high concentrations. A number of clinically
for the drug-metabolizing CYP3A enzymes.
used drugs such as the antibiotic rifampicin
The CYP3A enzymes are responsible for the
metabolism of almost 60% of all clinically
used drugs (Lehman et al. 1998). This makes
PXR very interesting since it is crucial to
progesterone and is included in the steroid
understand the mechanism underlying CYP3A
receptor superfamily. PXR has 2 isoforms:
PXR Influences Endocrine Therapies in Breast Cancer Materials and Methods Plasmid Prep of RSV β-gal Transfection
CV-1 cells were plated out on 24-well plates
1.2x105 cells/well. The transfection mixes
RNA prep from BK MCF-7, SF MCF-7,
contained 1 µl of Fugene 6, 33 ng receptor
ZR75-1 AND T47D
expression vector (hPXR), 100 ng of reporter
RNA was extracted from frozen cell pellet.
plasmid (CYP3A4), 356 ng of carrier plasmid
The cell pellet was dissolved in 700 µl
guanidium isothiocyanatate (GITC). 700 µl 2
expression vector/well. Cells were transfected
M NaAc pH 4.0, 700 µl phenol, 140 µl CHCl3
over night. The medium was changed to F12
were added. The solutions were centrifuged
Media with 10% delipidated serum containing
rifampicin, tamoxifen or 4-hydroxytamoxifen
at 7 different concentrations ranging from 0.1
solutions at –20°C for 1 hour. The solutions
nM to 100 µM. DMSO was used as control.
Cells were incubated over night. Cell extracts
rpm/15000xg). Pellet was dissolved in GITC
were prepared and assayed for luciferin and
solutions precipitated at –20°C for 1 hour.
Solutions were centrifuged (15 min, 4°C,
12000 rpm/15000xg). Pellet was washed with
PCR was carried out in a total of 25 µl,
ethanol (70%) and centrifuged (5 min, 4°C,
containing 2 µl of cDNA/RNA plasmid, 0.5
12000 rpm/15000xg). Pellet was air dried for
mM dNTPs, buffer, 10 pmol PXR nest 3`, 10
15 min at room temp then dissolved in 100 µl
pmol PXR nest 5`,10 pmol β-actin 5`, pmol
A regime of 95°C for 5 minutes followed by
cDNA prep from BK MCF-7, SF MCF-7,
30 cycles of 94°C for 1 min, 50°C for 1 min,
ZR75-1 AND T47D
72°C for 1.5 min and finished with 72°C for 7
min was used. PCR products were separated
inhibitor were added and the solutions were
on an agarose (1%) gel stained with Ethidium
made up to 10 µl with sterile filtered H2O.
Bromide. The primers used consisted of hPXR
The solutions were put at 37°C for 15 min,
65°C for 5 min. 1 µl oligo dT was added then
TCGGAGATCACCCGGAAGAC 3’; fragment size
put at 70°C for 10 min, put on ice to cool. 4
µl superscript buffer, 2 µl DTT and 2 µl 20 mM
dNTPs were added. Put at 42°C for 2 min.
1 µl superscript reverse transcriptase was
added. Put at 42°C for 50 min and finally put
PXR Influences Endocrine Therapies in Breast Cancer
was run as shown in fig 4. PXR was present
PXR is present in breast cancer cell lines
in BK MCF-7 DNA sample and absent in the
RT-PCR was used to determine whether PXR
was expressed any of the breast cancer cell
lines used. The cell lines that were used were
BK MCF-7, SF MCF-7, ZR75-1 and T47D. On the first agarose gel (1%) no signals were
seen. The conclusion was that the primers
could have interacted or that the amount of
PXR was too small to detect with agarose gel
Both 7) T47D RNA and 8) T47D DNA were visible which can’t be right
Polyacrylamide gel. The gel was analyzed by
since RNA can’t possibly bind to the PXR primers. 6) BK MCF-1 DNA contained PXR
indefinite. Another PCR (1) was set up and
and 5) BK MCF-7 RNA didn’t. That verifies
this time the primers were separated on a
that the BK MCF-7 cell line contain PXR.
contamination were encountered as shown in
SF RNA, 4, SF cDNA, 5,BK RNA, 6, BK cDNA, 7, T47D RNA, 8,T47D cDNA, 9,ZR75-1 RNA, Tamoxifen and 4-hydroxytamoxifen can activate PXR
activator of PXR. Rifampicin was therefore
hydroxytamoxifen and tamoxifen. Figure 5
Fig. 3 The agarose gel analysis indicated that there were PXR in almost all of the (1) samples except water suggesting that there had to be a contamination. Templates: 1,H2O, 2,HepG2 RNA, 3,HepG2 cDNA, 4,LMCAT RNA, 5,LMCAT cDNA, 6,BK RNA, 7,BK cDNA, 8,SF RNA, 9,SF cDNA, 10, T47D RNA, 11,T47D cDNA, 12,ZR75 RNA, 13,ZR75 cDNA, 14,PXR plasmid (1/10).
We used agarose gel analysis to find out which compound/s were contaminated. We
replaced H2O, RNAse inhibitor, dNTPS, superscript buffer and DTT. A new RT-PCR
PXR Influences Endocrine Therapies in Breast Cancer
Dose response curves of rifampicin, both
Discussion
The data presented here supports the theory
run. The first two transfections failed. A third
that tamoxifen and 4-hydroxytamoxifen can
activate PXR in vitro. The required
Rifampcin induced a 9 fold induction at 10
concentration, 10 µM, is however high. The
µM, 4-hydroxy tamoxifen gave a 6.2 fold
results imply that BK MCF-7 breast cancer
cell lines contain PXR and that it is active.
More tests need to be run before any final
considered activation. More tests need to be
conclusions can be drawn. What is to be done
run before any final conclusions and statistics
next is to look for the CYP3A enzyme in the
can be made. However it looks as if both
cell line and see if it is actively metabolizing
tamoxifen. Another task is to see if the
upgraded. The tests need to be repeated on
PXR/CYP3A regulated pathway works in vivo.
One essential goal is to determine if PXR is
Cells were analyzed for luciferase and ß-gal
involved in tamoxifen resistance. It is crucial
activity and the data represent the mean of 3
behind drug interaction and enhance the drug
folded induction at 10 µM yet tamoxifen and
4-hydroxy- tamoxifen also gave good inductions at 10 µM. 4-hydroxytamoxifen gave a 6.2 fold induction and tamoxifen gave a 4.4 fold induction.
PXR is present in the BK MCF-7 breast cancer cell line and it is active The cells were transfected and treated with
rifampicin, 4-hydroxytamoxifen and tamoxifen. Rifampicin and PXR was used as a positive control. Rifampicin activated PXR in
the BK MCF-7 breast cancer cell. This suggests that PXR is not only present in the
cell line, it is also active, as shown in diagram2.
PXR Influences Endocrine Therapies in Breast Cancer References
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Juha Niemelä Arthur Arkadius Kylander – Amerikansuomalainen kupletisti Artikkeli amerikansuomalaisesta lauluntekijästä Arthur Kylanderista Siirtolaisuusinstituutti – Migrationsinstitutet Turku – Åbo 2003 Arthur Arkadius Kylander – Amerikansuomalainen kupletisti Amerikansuomalainen lauluntekijä Arthur Arkadius Kylander syntyi Liedossa vuonna 1892 ja k
IUD (spirale) Lo IUD, più comunemente chiamato spirale, è un dispositivo anticoncezionale di tipo meccanico che viene inserito nell’utero. Questo dispositivo è formato da materiale plastico su cui è avvolto un filo di rame ed è di forma varia, lungo circa 4 centimetri e pesa pochi grammi. Il suo meccanismo di azione appare collegato a modeste modificazioni locali della mucosa uter