Effects of Drug Resistance on Skin Cancer

Emily Wang, Abbey Michaelson, Emma Johnson, and Colin O'Malley

Multidrug Resistant Cancers

Many cancers adapt to become multidrug-resistant (MDR), posing significant challenges in treatment design
Multidrug resistance (MDR) in cancer occurs through a variety of mechanisms including elevated drug efflux, reduced drug influx and changes in metabolism1
In response to external chemical cues cancer cells undergo metabolic plasticity, a factor that contributes to the development of the MDR phenotype2
- One notable example of metabolic plasticity is the Warburg Effect
It is unclear how the cancer cells are taking in our prodrugs
- If sugar transporters—e.g. glucose transporters (GLUTs)—are overexpressed in MDR cancers, we can tailor the prodrug’s sugar group to that transporter = optimize for ↑influx
Efflux pumps, such as P-glycoprotein (P-gp), are often overexpressed in MDR cancers and work to expel chemotherapeutics out of the cell3

Warburg Effect and GLUTs

Cancer cells favor glycolysis over oxidative phosphorylation4
Many cancer cells overexpress various GLUT isoforms
GLUTs are associated with glucose uptake, increase in GLUT expression may lead to an increase in glucose uptake5
Although multidrug resistant cells are well studied, there are no studies characterizing an entire chemoresistant gradient
Here, we characterize melanoma cells as they acquire resistance to doxorubicin (DOX), focusing on changes in GLUTs and glucose metabolism (Figure 2)

Methods

MDR Development

The murine melanoma B16-F10-Luc2 (B16) cell line was used as the in vitro cancer model
Concentration of DOX was doubled at each stable passage, from 1 nM to 1 μM

Cell Viability

B16 cells across 0-1000 nM resistance levels were grown in PBS or 5 μM DOX for 72 h to test resistance capacity. Resazurin assay was used to measure cell viability (Figure 4)

Western Blot

Cell lysates were extracted from B16 cells at various resistance levels and analyzed for GLUT1–4 and P-gp protein expression (Figure 5)

Glucose Uptake

Parent and 1 μM-resistant B16 were glucose-starved and fed varying concentrations of 2-NBDG to track glucose uptake. Fluorescence was measured by flow cytometry (Figures 8 and 10)

CSFE

Parent and 1 μM-resistant B16 were cultured in a 6-well plate with and without DOX and with and without CFSE. After 48 hours of incubation, samples were analyzed with a flow cytometer.

Luciferin

Parent and 1 μM-resistant B16 were treated with a 1 mg/mL Luciferin solution. After a 15-minute incubation, samples were analyzed with a flow cytometer (Figure 9)

Results

Image shows phenotypes of the Parent B16 cells and the multidrug resistant B16 cells. Parent cells appear spindle-shaped while resistant cells show dark clumps

Figure shows viability of the parent and multidrug resistant cells

Figure 1.

B16 phenotype changes in MDR

Parent cells appear spindle-shaped while MDR cells show differences in shape and color.

Figure 2.

Cell viability of B16 samples -/+ 5 μM DOX

Viability measured by Resazurin assay.

*P < 0.05; **P < 0.005

MDR cells show higher viability than parent cells.

Figure shows results of western blot ran for detection of P-gp and GLUT expression

Figure 3.

Western blot detection of P-gp/GLUT

(A) Without insulin

(B) With insulin for 3 or 24 h (to test effects on GLUT4)

P-gp expression appears to increase with an increase in MDR development. GLUT4 expression high in 1 uM resistant cells.

Graph shows glucose uptake of both parent and multidrug resistant cells at differing glucose concentrations

Figure 4.

Glucose uptake using 2-NBDG and flow cytometry

Figure 5.

CSFE done on B16 cells with both Dox and dye

Increased fluorescence with 1 μM resistance, DOX treatment, and CFSE dye. No generations observed.

Two graphs showing results of the CSFE assay done on parent B16 and multidrug resistant B16 cells, with and without doxorubicin and dye

Graph shows results of the luciferase assay done on parent and drug resistant B16 cells

Figure 6.

Luciferin was done on B16 parent cells and 1 uM MDR cells

No apparent change in Luciferin expression is observed between the parent and MDR cells.

Graph shows results of glucose uptake assay done on parent and drug resistant B16 cells after being fed high glucose media

Figure 7.

Glucose uptake using 2-NBDG and flow cytometry

Potential difference in glucose uptake between parent cells and MDR after being fed 8 g/L glucose media.

Conclusions

High expression of GLUT4 in 1 uM resistant cells
In vitro dye concentrations change with cell type and resistance level
No apparent change in luciferin expression between parent and MDR cells
Potential differences in glucose uptake between parent and MDR cells after being fed 8 g/L media

Future Research

Repeat the glucose uptake assay on the entire gradient
Modify the CFSE protocol
Glycolysis/respiration assays

In vivo studies

References

Emran, T. B., Shahriar, A., Mahmud, A. R., Rahman, T., Abir, M. H., Siddiquee, Mohd. F., Ahmed, H., Rahman, N., Nainu, F., Wahyudin, E., Mitra, S., Dhama, K., Habiballah, M. M., Haque, S., Islam, A., & Hassan, M. M. (2022). Multidrug resistance in cancer: Understanding molecular mechanisms, immunoprevention and therapeutic approaches. Frontiers in Oncology, 12. https://doi.org/10.3389/fonc.2022.891652

2. Bhat, G. R., Sethi, I., Sadida, H. Q., Rah, B., Mir, R., Algehainy, N., Albalawi, I. A., Masoodi, T., Subbaraj, G. K., Jamal, F., Singh, M., Kumar, R., Macha, M. A., Uddin, S., Akil, A. S., Haris, M., & Bhat, A. A. (2024). Cancer cell plasticity: From cellular, molecular, and genetic mechanisms to tumor heterogeneity and drug resistance. Cancer and Metastasis Reviews, 43(1), 197–228. https://doi.org/10.1007/s10555-024-10172-z

3. Gottesman, M. M., & Pastan, I. H. (2015). The role of multidrug resistance efflux pumps in cancer: Revisiting a JNCI publication exploring expression of the MDR1 (P-glycoprotein) gene. Journal of the National Cancer Institute, 107(9). https://doi.org/10.1093/jnci/djv222

4. Liberti, M. V., & Locasale, J. W. (2016). The Warburg effect: How does it benefit cancer cells? Trends in Biochemical Sciences, 41(3), 211–218. https://doi.org/10.1016/j.tibs.2015.12.001

5. Wang, T., Wang, J., Hu, X., Huang, X., & Chen, G.-X. (2020). Current understanding of glucose transporter 4 expression and functional mechanisms. World Journal of Biological Chemistry, 11(3), 76–98. https://doi.org/10.4331/wjbc.v11.i3.76

NACE Competencies

Career and Self-Development

Voluntarily participating in research with the Chemistry Department.

Communication

Demonstrating verbal and written abilities, communicating within a team for cell culture needs.

Teamwork

Building strong relationships with labmates throughout the semester while working together.

Technology

Navigating new technology for data display and analysis.

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