Formaldehyde increases the sensitivity of breast and ovarian cancer cells to chemotherapeutic drugs like doxorubicin, cisplatin, and 5-fluorouracil in a BRCA1/2-dependent manner. Experiments showed a synergistic growth inhibition effect when formaldehyde was combined with these drugs at low doses in BRCA1/2 deficient cell lines, but not in BRCA1/2 proficient cell lines. Further experiments indicated this synergistic response was due to increased DNA double-strand breaks and cell death, rather than just growth inhibition, when formaldehyde was combined with doxorubicin. The synergistic, cytotoxic response to formaldehyde combinations was also observed in BRCA1/2 deficient ovarian cancer cell

1. Formaldehyde Increases Sensitivity of Breast and Ovarian Cancer
Cells to Chemotherapeutic Drugs
Efren Alonso Cid1,2*, Jaeda Patton1,4*, Anuradha Kumari1, Amanda K. McCullough1,2,3
1Center for Research on Occupational and Environmental Toxicology, 2Knight Cancer Institute, 3Department of Molecular & Medical Genetics
Oregon Health & Science University, Portland, OR
4Cornell University, Ithaca, NY
*Both authors contributed equally to this poster.
Results
Table 1. Individual and combined response of BRCA-proficient and
-deficient cell lines to formaldehyde and other chemotherapeutic
drugs. Data was measured by CTG assay and is expressed as
percent growth inhibition. Concentrations are 40 μM (†60 μM) for
formaldehyde, 100 nM for doxorubicin, 20 ng/ml for mitomycin C,
150 nM for cisplatin, 6 μM for 5-fluorouracil, and 5 nM for
paclitaxel. Each value is an average of 3–6 independent values.
*Negative values are reported as 0.
References
1. National Toxicology Program. (2011). Formaldehyde. Report on Carcinogens, Twelfth
Edition. Retrieved from http://ntp.niehs.nih.gov/?objectid=03C9AF75-E1BF-FF40-
DBA9EC0928DF8B15
2. Swenberg, J. A., Lu, K., Moeller, B. C., Gao, L., Upton, P. B., Nakamura, J., & Starr, T. B.
(2011). Endogenous versus Exogenous DNA Adducts: Their Role in Carcinogenesis,
Epidemiology, and Risk Assessment. Toxicological Sciences, 120 (S1), S130–S145.
3. Barker, S., Weinfeld, M., & Murray, D. (2005). DNA-protein crosslinks: their induction, repair,
and biological consequences. Mutation Research/Reviews in Mutation Research, 589, 111–
135.
4. Kumari, A., Lim, Y. X., Newell, A. H., Olson, S. B., & McCullough, A. K. (2012).
Formaldehyde-induced genome instability is suppressed by an XPF-dependent pathway. DNA
Repair, 11, 236–246.
5. Nakano, T., Katafuchi, A., Matsubara, M., Terato, H., Tsuboi, T., Masuda, T., … Iijima, K.
(2009). Homologous Recombination but Not Nucleotide Excision Repair Plays a Pivotal Role in
Tolerance of DNA-Protein Cross-links in Mammalian Cells. The Journal of Biological
Chemistry, 284, 27065–27076.
6. Ridpath, J. R., Nakamura, A., Tano, K., Luke, A. M., Sonoda, E., … Nakamura, J. (2007).
Cells Deficient in the FANC/BRCA Pathway Are Hypersensitive to Plasma Levels of
Formaldehyde. Cancer Research, 67, 11117–11122.
7. Chakravarty, P. (2011). Use of formaldehyde as a novel agent for cancer therapy.
International Research Journal of Biotechnology, 2, 93–102.
8. Cutts, S. M., Rephaeli, A., Nudelman, A., Hmelnitsky, I., & Phillips, D. R. (2001). Molecular
Basis for the Synergistic Interaction of Adriamycin with the Formaldehyde-releasing Prodrug
Pivaloyloxymethyl Butyrate (AN-9). Cancer Research, 61, 8194–8202.
9. Swift, L. P., Rephaeli, A., Nudelman, A., Phillips, D. R., & Cutts, S. M. (2006). Doxorubicin-
DNA Adducts Induce a Non-Topoisomerase II-Mediated Form of Cell Death. Cancer Research,
66, 4863–4871.
10. Roy, R., Chun, J., & Powell, S. N. (2012). BRCA1 and BRCA2: different roles in a common
pathway of genome protection. Nature Reviews. Cancer, 12, 68–78.
11. Caestecker, K. W. & Van de Walle, G. R. (2013). The role of BRCA1 in DNA double-strand
repair: Past and present. Experimental Cell Research, 319, 575–587.
Acknowledgements
MCF7 and HCC1937 cell lines were a gift from Mushui Dai and Rosalie Sears (OHSU).
We thank OHSU Knight Cancer Institute’s CURE program and the CROET summer
internship program for funding the internships that this project was a part of. This
research is filed under Invention Disclosure [ID #1758].
Funding for this research is provided by the NIH National Cancer Institute ROI CA
106858 (AKM).
Drugs MCF7 HCC1937 HCC1395
UWB1.289
+BRCA1
UWB1.289
Formaldehyde 28.3 2.9 4.0 46.0 40.4
Doxorubicin 47.1 3.3 0* 31.2 0*
Formaldehyde x Doxorubicin 83.7 87.1 62.4 89.0 83.4
Formaldehyde 8.5 2.8 - 35.5 52.7
Mitomycin C 28.5 4.7 - 15.8 0*
Formaldehyde x Mitomycin C 40.8 18.4 - 33.1 46.7
Formaldehyde - - - 32.4 47.4
Cisplatin - - - 3.8 0*
Formaldehyde x Cisplatin - - - 28.6 52.4
Formaldehyde 25.2† 6.7†
- 55.1 39.6
5-Fluorouracil 43.4 2.8 - 44.9 8.2
Formaldehyde x 5-Fluorouracil 53.4† 48.3†
- 54.1 41.7
Formaldehyde 24.6 20.8 - 21.2 57.9
Paclitaxel 31.0 24.2 - 0* 38.1
Formaldehyde x Paclitaxel 37.2 48.5 - 31.5 55.9
Conclusions
• In agreement with our hypothesis, the BRCA1/2-deficient cell
lines were more sensitive to combined formaldehyde
treatments than proficient cell lines.
• Some combinations produced a synergistic growth inhibition
effect at doses that individually yielded low cytotoxicity.
• Based on imaging data, the drugs were indeed killing the cells,
rather than simply inhibiting growth.
• The formaldehyde concentrations used in this study are within
the range that is found endogenously in the body, and are
therefore easy for the body to metabolize.
• Since drugs act synergistically with formaldehyde, they can be
administered to patients at low doses, minimizing side effects.
Results
Figure 1. Breast cancer cell growth inhibition from individual and combined formaldehyde and
doxorubicin treatments. Growth inhibition was measured by CTG assay and relative
luminescence was normalized to the control. Formaldehyde (F) concentration is measured in
μM and doxorubicin (D) concentration is measured in nM, with concentration increasing
along the x-axis.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
RelativeLuminescence
Drug Concentration
HCC1395
Formaldehyde (10-40)
Doxorubicin (50-200)
F20XD (50-200)
F40XD (50-200)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
RelativeLuminescence
Drug Concentration
HCC1937
Formaldehyde (20-60)
Doxorubicin (50-200)
F20XD (50-200)
F40XD (50-200)
B C
0
0.2
0.4
0.6
0.8
1
1.2
1.4
RelativeLuminescence
Drug Concentration
MCF7
Formaldehyde (20-60)
Doxorubicin (50-200)
F20XD (50-200)
F40XD (50-200)
A
0
0.2
0.4
0.6
0.8
1
1.2
1.4
RelativeLuminescence
Drug Concentration
UWB1.289+BRCA1
Formaldehyde (10-40)
Doxorubicin (50-200)
F20XD (50-200)
F40XD (50-200)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
RelativeLuminescence
Drug Concentration
UWB1.289
Formaldehyde (10-40)
Doxorubicin (50-200)
F20XD (50-200)
F40XD (50-200)
BA
Figure 2. Ovarian cancer cell growth inhibition from individual and combined formaldehyde
and doxorubicin treatments. Assay and data presentation same as in Figure 1.
Synergistic response to several drugs in combination
with formaldehyde in breast cancer cells in a BRCA1/2-
dependent manner
In order to examine whether formaldehyde could enhance
the efficacy of known anti-cancer drugs, we studied the
cytotoxicity of several commonly used anti-cancer drugs (5-
fluorouracil, cisplatin, mitomycin C, paclitaxel, and
doxorubicin) on a HR-proficient (MCF7) and a HR-deficient
(HCC1937, BRCA1-deficient) breast cancer cell line. A dose
range yielding low cytotoxicity (10–20%) was chosen for all
drugs, including formaldehyde. In contrast to the BRCA1-
proficient breast cancer cell line (MCF7), a more pronounced
and synergistic response was observed for all the
combinations in BRCA1-deficient HCC1937 cells (Table 1).
Among all the combinations tested, the most striking
response was observed with the doxorubicin-formaldehyde
combination (Figure 1A and B).
To further explore the possibility that formaldehyde-induced
synergism was not limited to BRCA1 but could be expanded
to the other HR-related genes, we studied the effect of
doxorubicin and formaldehyde on a BRCA2-deficient breast
cancer cell line. Consistent with the results obtained with
BRCA1-deficient breast cancer cells, BRCA2-deficient breast
cancer cells exhibited a synergistic response to the
doxorubicin-formaldehyde combination (Table 1, Figure 1C).
Other combinations have yet to be tested on this cell line.
Synergistic response to doxorubicin in combination with
formaldehyde in breast cancer cells is expandable to
other types of cancer
To investigate whether BRCA1-dependency is confined to
breast cancer cell lines, we studied the effects of
combination treatments on BRCA1-deficient and -proficient
ovarian cancer cell lines. Analogous to other repair-deficient
cell lines, the formaldehyde-doxorubicin resulted in a similar
synergistic response in BRCA1-deficient cells relative to
BRCA1-complemented ovarian cancer cells (Figure 2A and
B). Synergism was also observed in the BRCA1-deficient
cells in response to formaldehyde and cisplatin, but not with
other combinations (Table 1). Synergism was determined to
be statistically significant for high formaldehyde and
doxorubicin concentrations (data not shown). We are in the
process of performing statistical analysis on other cell lines
and drug combinations.
Accumulation of DNA DSBs following doxorubicin–
formaldehyde combination
An accumulation of double-strand breaks (DSBs) in response
to combined treatments of doxorubicin and formaldehyde
was observed in both MCF7 and HCC1937 cells (Figure 3).
Additionally, accumulation of giant nuclei (Figure 4A and B
(circled)) was observed in both MCF7 and HCC1937 cells
post-formaldehyde and doxorubicin combined treatments.
Combined treatments with doxorubicin and
formaldehyde results in cell death, not growth inhibition
Due to the limitation of the CTG assay not being able to
distinguish between growth inhibition and cell death, we
looked at the morphology of MCF7 and HCC1937 cells
following combined treatments with doxorubicin and
formaldehyde. Consistent with the CTG assays, relative to
MCF7 cells, increased cell death (based on DAPI staining)
was observed in HCC1937 cells following a combined
treatment with doxorubicin and formaldehyde (Figure 4A and
B (arrows), C).
More direct assays are required to measure apoptotic
responses to the above-mentioned combined treatments. In
addition, the mechanism behind these responses remains to
be determined.
MCF7HCC1937
Figure 3. Accumulation of DNA double strand breaks (DSBs) for MCF7 and HCC1937. Blue
channel shows nuclear staining, while green channel shows 53BP1 foci, which form at DSB
sites (A, B). We counted the number of cells with ≥10 foci to account for DSBs that may have
been formed due to endogenous DNA damage, and not from the treatment. This endogenous
damage can be seen in the control cells, which received no treatment. Averages of 3
independent experiments are shown, with error bars representing standard deviation (C).
Control F20XD50 F20XD100
0
20
40
60
80
100
Control F20XD50 F20XD100
Cellswith≥10Foci(%)
MCF7 HCC1937
C
A
B
0
10
20
30
40
50
Control F20XD50 F20XD100
ApoptoticCells(%)
MCF7 HCC1937
C
Figure 4. Apoptosis and giant nuclei formation in MCF7 and HCC1937. Blue channel shows
nuclear staining. Giant nuclei are circled; arrows point to apoptotic cells (A, B). Cells were
counted as apoptotic if their nuclei were fragmented. Averages of 3 independent experiments
are shown, with error bars representing standard deviation (C).
MCF7HCC1937
A
B
Control F20XD50 F20XD100
Formaldehyde is a common environmental and occupational
chemical and is classified as a known human carcinogen (1). The
human body contains endogenous formaldehyde—about 100 μM
in the blood—which is quickly and easily metabolized (2), but
higher concentrations are known to cause DNA damage,
especially in the form of DNA-protein crosslinks (DPCs) (3).
Studies have shown that homologous recombination (HR), and to
a lesser extent nucleotide excision repair (NER), are the most
important pathways involved in repairing formaldehyde-induced
DPCs in mammalian cells (4–6). Some cancers, especially breast
and ovarian cancer, can be caused by mutations in HR repair
pathways—thus, there exists the possibility that these cancer
types could be targeted for treatment using formaldehyde to
overwhelm the HR pathway. Although the chemotherapeutic
potential of formaldehyde both individually (7) and in the form of
formaldehyde-releasing prodrugs in combination with doxorubicin
(8, 9) has been investigated, its effectiveness has not been
previously tested on HR-deficient cells. BRCA1 and BRCA2 are
two HR-related genes that have been extensively studied since
their discovery as primary risk factors for hereditary breast and
ovarian cancer (HBOC) syndrome (10, 11). Mutations in both
genes have also been shown to incur hypersensitivity to
formaldehyde (5, 6), making them excellent targets for
formaldehyde treatment.
The aim of this study was to investigate whether formaldehyde in
combination with certain chemotherapeutic drugs with different
mechanisms of cytotoxicity produces a synergistic killing effect on
BRCA1- and BRCA2-deficient breast and ovarian cancer cells.
We hypothesized that BRCA-deficient cells would respond better
to treatment than BRCA-proficient cells, and that cells would be
killed at low drug concentrations in combination with
formaldehyde.
Introduction
Cells and culture conditions
Breast cancer cell lines MCF7 (BRCA1/2-proficient), HCC1937
(BRCA1-deficient), and HCC1395 (BRCA2-deficient) and ovarian
cancer cell lines UWB1.289+BRCA1 (BRCA1-complemented)
and UWB1.289 (BRCA1-deficient) (ATCC) were used in this
study. Cells were cultured according to manufacturers’
suggestions.
Growth inhibition assays
Growth inhibition was measured using the CellTiter-Glo
Luminescent Cell Viability (CTG) Assay (Promega). Cells were
seeded in 96-well plates and allowed to attach. Drugs were
administered at indicated concentrations and incubated at 37°C
for 5 days (MCF7 and HCC1937) or 3 days (all others). CTG
assays were performed according to manufacturer’s instructions.
Immunofluorescence staining
Immunofluorescence staining was used to measure 53BP1 foci
formation and apoptosis. Cells were seeded onto coverslips and
treated for 3 days at indicated drug concentrations. After fixation,
permeabilization, and blocking, cells were incubated with anti-
human 53BP1 (1:1000 IN PBS) and Alexa Fluor anti-mouse 488
(1:500 in PBS) for 53BP1 foci staining, and 1 μl DAPI (0.5 mg/ml)
in 1 ml PBS for nuclear staining. Coverslips were fixed onto slides
and visualized at 40X objective using a Zeiss microscope.
Materials and Methods