1. Tessa Witkowsky , Ashok Singh, and Ajit Kumar VermaTessa Witkowsky , Ashok Singh, and Ajit Kumar Verma
Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin Medical School,Department of Human Oncology, School of Medicine and Public Health, University of Wisconsin Medical School,
Madison, WI, 53705Madison, WI, 53705
The Prostate gland is dependent on androgen (testosterone and
dihydrotestosterone (DHT)) via the androgen receptor (AR) signaling
pathway. Uncontrollable cell growth, due to increased levels of prostate-
specific antigens (PSA), is the main cause of Prostate cancer (Armstrong et
al., 2014). Initial treatments include androgen deprivation therapies
(ADT), which lower the levels of androgen present in the Prostate.
However, resistance to ADT often occurs as cancer progresses to CRPC.
CRPC is a aggressive and it is this stage at which most deaths due to PCa
occur.
Hypothesis
To determine whether MDR1 over-expression is linked to ENZ resistance in
22Rv1 PCa cells. Based on previous research, 22Rv1 ENZ treated cells
will express MDR1 and will therefore be coined resistant.
Prostate Cancer (PCa) is one of the leading causes of death in males across
the world. PCa is a result of uncontrollable cell growth due to increased
levels of prostate-specific antigens (PSA). Initial treatments include
androgen-deprivation therapies but the cancer will ultimately progress to
CRPC (Armstrong et al., 2014). ENZ inhibits the androgen-receptor
signaling pathway at 3 key stages and is a known treatment to CRPC. Over
expression of MDR1 has been correlated with drug resistance in various
malignant tumor cells (Finch and Pilians, 2014). We are investigating
whether MDR1 over-expression in Enzalutamide (ENZ)-resistant 22Rv1
PCa cells is a contributor to drug resistance in CRPC. The results will
provide preclinical evidence he control of CRPC.
The rate of PCa incidence
and mortality in the
world.
Thanks to Dr. Ajit Verma, funded by NIH grant CA102431, for facilitating this study. Finally thanks to
my senior student mentor Satyamedha Bathula for assisting me with lab techniques and
Saivenkateshkomal Bathula for supporting me.
 Microscopy images show no external morphological differences between
22Rv1 parental and 22Rv1-R. Looking at the 20x magnification, there
seems to be a greater proportion of some substance (ribosomes/proteins) in
22Rv1-R than in 22Rv1 parental.
Western analysis show 22Rv1-R express MDR1 at a higher concentration
than 22Rv1 parental
MTT shows cell viability remaining relatively constant for 22RV1-R while
continuing to decrease for 22Rv1 parental as the concentration of ENZ
increases.
22Rv1-R
MDR1
22Rv1
MDR1
MDR1 expression in 22Rv1 parental and 22Rv1-R cells
After 10 treatments of 15 uM ENZ, it was concluded that 22Rv1-R cells had
developed resistance to Enzalutamide. 22Rv1-R cells expressed MDR1 at a
higher concentration than 22Rv1 parental cells. 22Rv1-R cell viability
remained stable as the dose of ENZ increased while 22Rv1 parental cell
viability continued to decrease. Stability of cell viability in various
concentrations of drug indicates resistance has been achieved. Sufficient
evidence has been provided to support the hypothesis of MDR1 over-
expression being linked to ENZ resistance in 22Rv1 PCa cells.
ENZ is an approved drug for CRPC that inhibits the AR signaling pathway
at three stages: blocking the binding of androgens to AR, inhibiting
nuclear translocation of activated AR, and impairing binding of activated
AR with DNA (Fitzpatrick and Schalken, 2015). Based on previous
research, it was concluded that MDR1 over-expression is correlated with
drug resistant tumor cells (Kazuhiro and Yoshikazu, 2014). P-glycoprotein
is an efflux pump that pumps foreign substances out of the cell. Over
expression of MDR1 in tumor cells reduces the bioavailability of cytotoxic
drugs which leads to resistance.
MDR1
Actin
22Rv1
22Rv1-R
A.N: 0.0 1.2
MDR1 is expressed in 22RV1 – Docetaxel (DTX) resistant cells but
not in 22RV1 control cells. DTX is an approved drug for metastatic
PCa.
Pca cell line: 22Rv1 is used in order to generate 22Rv1-R (Enzalutamide Resistant
cells).
Drugs used: Enzalutamide (ENZ)
Generation of ENZ resistant 22Rv1 cells (22Rv1-R): 22Rv1 cell lines were
treated with a 15 Mμ dose concentration of ENZ for 10 consecutive treatments.
Protein Lysate: To procure the protein only in parental and resistant cells.
Protein Estimation: To determine the relative concentrations of protein present in
each cell line in order to obtain accurate reliable results from the Western Blot.
Western blotting: For biochemical analysis of MDR1 protein expression in 22Rv1
control and 22Rv1-R cells.
 MTT assay: To measure the cell viability/growth of 22Rv1 parental and 22Rv1
cells when treated with different concentrations of ENZ.
22Rv1-parental (20x)22Rv1-parental (10x)
22Rv1-R (10x)
Introduction
Abstract Experimental Methods
Characterization of ENZ resistant cells
After 10 treatments of 15 Mμ
ENZ there are no apparent
exterior morphological
differences between 22Rv1
control/parental and 22Rv1- ENZ
resistant cells when looked at
under a 10x magnification
microscope. However, when
looked at under a 20x
magnification, 22Rv1-resistant
cells seem to have a greater
proportion of ribosomes/protein.
22Rv1-R (20x)
MTT Assay
Enzalutamide Mechanism of Action
MTT (3-[4,5-dimethylthiazol-2-
yl]-2,5 diphenyl tetrazolium
bromid) assay measures the
amount of MTT absorbed by
living cells. MTT absorbance is
detected via the conversion of the
MTT molecule into formazan
crystals by the mitochondrial
activity of living cells and is
measured using Optical Density
at 450 nm. Lower percent
OD450 absorbance means there
are less number of viable cells.
(i.e. more cell death at that drug
concentration)
Results
Conclusion
References
Acknowledgments
1. Tran C, et al. Science 2009;324:787–90. 2. Hu R, et al. Expert Rev Endocrinol Metab 2010;5:753–64.
2. Microscopy images show no external morphological differences between 22Rv1 parental and 22Rv1-R.
3. Armstrong A., Beer T., Bono J., Bhattacharya S., Carles J., Chowdhury S., Davis I., Evans C., Fizazi K., Kim C., Kimura G., Higano C., Iverson
P., Joshua A., Loriot Y., Mainwaring P., Mansbach H., Miller K., Noonber S., Perabo F., Phung D., Rathkopf D., Saad F., Scher H., Sternberg C.,
Taplin M., Tombal B., and Vener P. 2014. Enzalutamide in Metastatic Prostate Cancer before Chemotherapy. The New England Journal of
Medicine. 371: 424-428.
4. Finch A., and Pilians P. 2014. P-glycoprotein and its role in drug-drug interactions. Australian Prescriber. 37: 137-138.
5. Kazuhiro K., Kohji N., and Yoshikazu S. 2014. Regulations of P-Glycoprotein/ABCB1/MDR1 in Human Cancer Cells. New Journal of Science.
2014: 1-2.
Enzalutamide mechanism of action in Pca patients with
initial progression to CRPC. Parental cells non-resistant
to ENZ: 22Rv1 parental
Ref. 1
Enzalutamide drug molecule
Actin
+ivecontrol
MDR1
ENZ 22Rv1-R Parental
MDR1 expression in 22Rv1-R
and 22Rv1 Parental cell lines