Presented at the STLCOP Research Symposium in the Spring of 2015.

1. Benjamin J. Minch, Pharm D. Candidate 2017
Nicholas R. Potter, Pharm D. Candidate 2017
Mentors: Noha N. Salama, B.Sc. Pharm. Ph.D.
Abdel Kareem Azab, B.Sc. Pharm Ph.D.
April 18th , 2015

2.  Multiple Myeloma (MM) is cancer of the bone
marrow caused by malignant plasma cells.
 Characterized by:
▪ Low RBC levels, Infection, Bone fractures
 Diagnosed by biopsy
 The second most common hematological
malignancy which remains incurable.
 There are 20,000 new cases and 10,000 deaths.
 Median survival of 4-5 years; recently extended to 7
due to advances in therapy.
http://www.cancer.org/cancer/multiplemyeloma/detailedguide/multiple-myeloma-risk-factors

3. http://www.mdpi.com/2227-7382/1/2/87
https://www.bikaken.or.jp/english/laboratories/numazu/summary.html

4.  Traditional Chemotherapy
 Doxorubicin (Adriamycin®)
 Proteasome inhibitors
 Bortezomib (Velcade®)
 Carfilzomib (Kyprolis® )
 Corticosteroids
 Bisphosphonates
 Zoledronic acid (Zometa®).

5.  P-glycoprotein(P-gp) plays a major role in the
development of multidrug resistance.
 Most extensively studied ATPase-Binding Cassette
transporter (ABC).
 Broad substrate specificity including Bortezomib
(Velcade®) and Doxorubicin (Adriamycin®).
 P-gp expression is increased by 50% in MM
patients after initiation of treatment.
 Hypoxia is believed to be an inducer of P-gp
expression.

6. We hypothesize that P-gp is upregulated in MM
cells under hypoxia which contributes to the
development of drug resistance.
Study Objective: Examine the effect of hypoxia on the
intracellular accumulation of Rhodamine 123 (Rh123, a
model P-gp substrate) in MM cell lines.

7.  Intracellular substrate accumulation is a standard
indirect approach to measure P-gp activity.
 Rhodamine 123 (Rh123) is a fluorescent P-gp
substrate.
 Preliminary studies: Incubation of the MM cell
line RPMI 8226 (B-lymphocyte) with Rh123
under hypoxic vs. normoxic conditions.
 1% O2 for hypoxia and 21% O2 for normoxia.
 Different Rh123 concentrations.
 Different incubation time points.

8.  Subsequent studies tested Rh123 efflux from
MM cell lines
 MM1S, H929, OMP2, U266
 N=3/treatment/cell line
 Analyzed via flow cytometry.
 ANOVA and Bonferroni pairwise comparisons.

9.  Most prominent Rh123 efflux from RPMI cells
under hypoxia at 1uM after 1 hr. incubation
44.1
60.8
77.3
68.1
28.3
33.2
14.5
41.8
31.7 32.1
14.6
39.4
28.0
36.0
17.3
38.138.9
34.3
12.7
61.9
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0.25 0.5 1 5
R123intracellularaccumulationin
hypoxiacomparedtoNormoxia
(PercentFluorescenceunits)
Rhodamine 123 concentration (uM)
0.5 hr
1 hr
3 hr
6 hr
24 hr
Incubation
times

10.  ~ 31% decrease in intracellular Rh123 accumulation
under hypoxia regardless of cell line (p < 0.001).
100.00
100.00 100.00 100.00
61.82
68.95 69.64
74.74
0.00
20.00
40.00
60.00
80.00
100.00
120.00
MM1s H929 OPM2 U266
Multiple Myeloma Cell Lines
Normoxia
Hypoxia
R123intracellularaccumulation
inhypoxiacomparedtoNormoxia
(PercentFluorescenceunits)
* P< 0.001
*
***

11.  Hypoxic conditions substantially increased the
extent of Rh123 efflux from all MM cell lines.
 Suggesting an increase in P-gp activity (expression
and/or functionality) in MM cell lines under hypoxia.
 Our findings present an opportunity to target
hypoxia in an attempt to interfere with the
development of P-gp-mediated drug resistance.
 Potential improvement in drug therapy and clinical
outcomes.

12.  Evaluate different therapeutic interventions
to combat hypoxic induction of P-gp and
reduce the incidence of refractory multiple
myeloma.
 Additional mechanistic studies may lead to
new therapeutic targets.

13. Noha N. Salama, B.Sc. Pharm. Ph.D.
Financial Support:
 Grant UL1TR000448
• Research reported in this poster (or presentation) was supported by the Washington
University Institute of Clinical and Translational Sciences grant UL1TR000448 from the
National Center for Advancing Translational Sciences (NCATS) of the National
Institutes of Health (NIH). The content is solely the responsibility of the authors and
does not necessarily represent the official view of the NIH.
 Federal work Study Funds
 Departmental Research Assistantship Funds
Barbara Muz, Ph.D.
Abdel Kareem Azab, B.Sc. Pharm Ph.D.