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Background: Although
differentiated thyroid cancer has good prognosis, radioactive iodine (RAI) resistant thyroid
cancer is difficult to treat. Current
therapies for progressive RAI resistant thyroid
cancer are not very effective. There is an unmet need for better therapeutic
agents in this scenario.
Studies have shown that aggressive thyroid cancers express matrix metalloproteinase -2
(MMP-2). Chlorotoxin is a selective MMP-2 agonist. Given that Saporin is a well-known
ribosome-inactivating protein used for anti-cancer treatment, we hypothesized that
Chlorotoxin-conjugated Saporin (CTX-SAP) would inhibit the growth of aggressive thyroid cancer cell lines
expressing MMP-2.
Methods: The ML-1
thyroid cancer cell line was used for this study because it is known to express MMP-2. ML-1 cells were
treated with a toxin consisting of biotinylated Chlorotoxin bonded with a secondary conjugate of
Streptavidin-ZAP containing Saporin (CTX-SAP) from 0 to 600 nm for 72 hours. Then,
cell viability was measured via XTT assay at an absorbance of A450-630. Control
experiments were set up using Chlorotoxin and Saporin individually at the same
varying concentrations.
Results: After 7 hours
of incubation, there was a statistically significant reduction in cell
viability with increasing concentrations of the CTX-SAP conjugate (F=4.286,
p=0.0057). In particular, the cell viability of ML-1 cells was decreased by
49.77% with the treatment of 600 nm of CTX-SAP (F=44.24), and the reduction in
cell viability was statistically significant (Dunnett’s test p<0.0001). In
contrast, individual Chlorotoxin or Saporin in increasing concentrations had no
significant effect on cell viability under the same conditions.
Conclusion: This in
vitro study demonstrated the efficacy of a CTX-SAP conjugate in reducing the
viability of ML-1 thyroid cancer cells in a dose dependent manner. Further
studies are needed to delineate the effectiveness of CTX-SAP in the treatment
of aggressive thyroid cancer. Our study points towards MMP-2 as a potential
target for RAI-resistant thyroid cancer.
Keywords:
Saporin, Chlorotoxin, Radioactive Iodine Resistance, Thyroid Cancer, MMP-2,
ML-1
INTRODUCTION
Incidence of thyroid cancer has been increasing in the past decade.
Estimated incidence of thyroid
cancer in 2019 is 52,070 [1]. It is predicted that by 2030, thyroid cancer will
be the fourth leading cause of new
cancer diagnosis in the United States [2]. In 2016 there were 822,242 patients living with thyroid cancer in the United
States [1]. Most of the thyroid cancers
respond well to surgery, radioactive iodine, and thyroid stimulating hormone (TSH) suppression. However, a subset of
these thyroid cancers will develop metastasis and become radioactive iodine (RAI) resistant. According to a
study by Schlumberger et al.,
up to 50% of thyroid cancer with metastasis may develop inability to
concentrate iodine [3]. When thyroid cancer cells become resistant to RAI,
newer therapeutic agents like tyrosine kinase
inhibitors could be used. Even with these newer therapeutic agents, most
RAI resistant metastatic thyroid cancer will progress. This could also result
in multiple toxicities. Even with newer therapeutic agents’ average progression
free interval is about 18 months [4]
and the ten-year mortality rate for these
kinds of thyroid cancers can reach up to 50% [5]. Hence, there is
an unmet
Chlorotoxin was initially identified in the venom of the scorpion Leiurus quinquestriatus [6]. It binds specifically to isoform
2 of matrix metalloproteinase (MMP-2) [7]. MMP-2 is overexpressed in aggressive thyroid cancers when compared
to normal thyroid tissue [8]. In Tumors
with larger size, extrathyroidal invasion and lymph node metastasis were found to overexpress MMP-2 [9]. Papillary
thyroid cancers (PTC) with lymph node metastasis were found to have significantly higher rates of pro-MMP-2
activation when compared to follicular
adenomas and normal thyroid tissue [10]. Widely invasive follicular thyroid
cancer was also found to express MMP-2 [11].
Saporin (SAP) is a ribosome-inactivating protein (RIP) derived from the
seeds of the soapwort plant, Saponaria officinalis [12]. The
mechanism of action of Saporin has been well studied and successfully employed
in the creation of immunotoxins. Saporin is very stable in vivo and is
resistant to proteases in the blood. Since Saporin works through many different
cell death pathways, it is hard to develop resistance to it [13-15]. Saporin by
itself is unable to cause
significant cell damage, as it cannot enter the cell efficiently on its own
[15]. Conjugation with antibodies or
other toxins which promotes its internalization, confers lethality to SAP. The
first study using an antibody conjugated with SAP was conducted in humans for
refractory Hodgkin’s disease, in which 75% of the patients achieved complete
remission and 50% of them
experienced relief from symptoms [16].
A recent study used Substance P conjugated with SAP
intrathecally in cancer patients with intractable pain [17].
ML-1 (ACC-464), thyroid cancer cell comes from dedifferentiated
recurrent follicular thyroid cancer
from a 50-year-old patient [18]. ML-1 is tumorigenic in rodents. Grimm et al.,
demonstrated that ML-1 cell
lines express MMP-2 [19]. In this
study we assessed
the effect of CTX-conjugated with SAP (CTX-SAP) on
cell viability of ML-1 cells.
MATERIALS AND METHODS
Toxins and
reagents
The Chlorotoxin-Saporin (BETA 010) conjugate was acquired from Advanced
Targeting Systems (San Diego, CA). This toxin consisted of biotinylated Chlorotoxin
bonded with a secondary conjugate of Streptavidin-ZAP containing Saporin. Unconjugated
Saporin and Chlorotoxin were acquired through Sigma-Aldrich. Dulbecco’s Modified
Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Phosphate Buffered Saline (PBS),
Trypsin-EDTA (and unconjugated Trypsin), Propidium Iodide (PI), and
Penicillin-Streptomycin antibodies were ordered from Fischer Scientific. XTT
activation reagent (PMS) and solution were purchased from Biotium.
Thyroid cancer
cell line
ML-1 (ACC-464)
thyroid cancer cells were acquired from the DSMZ German Leibniz Institute of Microorganisms and Cell Cultures. ML-1
cells were cultured in DMEM supplemented
with 10% FBS and 1% Penicillin-Streptomycin antibodies in a 75 cm3
Corning culture flask at 37°C and 5% CO2.
Toxin treatment
Cells were seeded at a density of 7500 cells/well on a 96-well plate
and given 24 h to incubate and attach to the 96-well plate. Then, varying
amounts of CTX, SAP, and CTX-SAP were treated in triplicate or quadruplet
repeats to the cells with an increasing dosage, ranging from 0 (NTC) to 600 nm,
by using 2 µM stock solutions for CTX, SAP, and CTX-SAP. The final volume of
media including the toxin treatment per each well was 100 µL. This was incubated
for a period of 72 h.
Cell viability
XTT(2,3-Bis-(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide,
disodium salt) assay is based off the cleavage of the tetrazolium salt. XTT, in
the presence of N-methyl dibenzopyrazine methyl sulfate (PMS), an
electron-acceptor, XTT is reduced to form a water soluble, orange-colored
formazan salt [20]. This type of reaction can only occur in viable,
metabolically-active cells, and therefore, the amount of dye formed is directly
related to the number of metabolically active cells present [21]. In this
study, after a 72-h incubation period with toxin treatment, 25 µL of activated
XTT dye was added to each well containing 7,500 cells according to the
manufacturer’s guidelines without removing media, and the level of color change
was quantified using a multi-well spectrophotometer (A450-630) [22]. This
absrobance was determined by the Biotium manufacturer. The protocol was to read
the plate at an absorbance of A450-500 nm, against the background of 630-690
nm. This is done by setting the plate-reading spectrophotometer to Delta.
Statistical
analysis
Prism8 statistical software (GraphPad Software Inc.) was utilized to
conduct statistical analysis. One-Way ANOVA was performed to compare absorbance
values between toxin-treated groups
and non-treated controls.
Post hoc comparisons were done using Dunnett’s test to compare values from
toxin-treated groups to the NTC. All values are expressed as the mean and
standard deviation of recorded absorbance values. P-values calculated from Dunnett’s test were adjusted to account for
multiple comparisons.
Unconjugated chlorotoxin (CTX)
ML-1 cells were exposed to unconjugated chlorotoxin at concentrations
ranging from 0 to 600 nm for
72 hours (Figure 1A). There was an overall statistically significant
difference in cell viability
assessed after 7 hours of incubation in the presence of XTT and PMS (F=3.34, p=0.038). The largest difference
was slightly increased viability (absorbance) of the cells exposed to 600 nm of unconjugated CTX
relative to the non-treated control (mean difference in absorbance - 0.054). However, this was not statistically significant with a Dunnett’s test
(p=0.1279).
Saporin alone (SAP)
ML-1 cells were exposed to unconjugated Saporin at concentrations
ranging from 0 to 600 nm (Figure
1B). There was an overall statistically significant difference in cell
viability after 7 hours of
incubation with XTT and PMS (F=3.271, p=0.0407). The largest difference was improved viability
(absorbance) of the cells exposed to 20 nm of unconjugated SAP relative to the non-treated control (mean
difference in absorbance -0.05325). However, this was not statistically significant with a Dunnett’s
test (p=0.0874).
Chlorotoxin-saporin conjugate (CTX-SAP)
ML-1
cells were exposed to Chlorotoxin-Saporin conjugate at concentrations ranging from 0 to 200 nm (Figure
2A). There was an overall statistically significant difference in cell viability at 7 hours of
incubation with XTT and PMS (F=4.286, p=0.0057) with an apparent trend for decreased viability with
increasing concentration of the conjugate. Post hoc statistical comparisons using Dunnett’s test showed no significantly reduced
viability for cells exposed to 2, 10 or 20 nm relative to non-treated control
(p>0.05). However, cells exposed to CTX-SAP
conjugate at concentrations of 40, 100 and 200 nm had significantly reduced
viability relative to non-treated controls
(Dunnett’s tests, p=0.0138, p=0.0052, and p=0.0037 for 40, 100 and 200 nm, respectively, relative to NTC).
DISCUSSION
This study
addressed the effectiveness of Saporin, Chlorotoxin and CTX-SAP conjugate in decreasing cell viability of
ML-1 cells. Saporin, a toxin from the plant seed Saponaria officinalis,
inhibits proliferation or cell viability of cancer cells
[23]. Previous studies have proven that Saporin is an effective ribosome-inactivating protein (RIP) that inhibits
protein synthesis and growth of both normal and tumor
cells [ 24].
However, our data
shows that there were no significant effects on ML-1 cancer cell
viability when treated with unconjugated Saporin up to 600 nm (Figure
1A). This would be in agreement with other studies that have chosen to use Saporin conjugates for cancer
therapies rather than unconjugated Saporin
alone [12]. This data
suggested the need for a vehicle that can be conjugated to Saporin and help
the toxin be internalized.
Chlorotoxin, which is a 36 amino-acid peptide from the venom of Leiurus
quinquestriatus, has been previously
used as a vehicle to deliver anti-cancer drugs to cancer cells [25].
Chlorotoxin is also a known MMP-2 isoform agonist, that makes an effective
vehicle for internalization as most aggressive thyroid cancers express MMP-2
receptors [7].
MMP-2 belongs to the matrix
metalloproteinases family which helps in degradation of basement membranes and extracellular matrix. MMP-2 is also
associated with angiogenesis inside
the tumor [26]. This promotes spread of the cancer. Activation of MMP-2 can
occur at the cell membrane. MMP-2
production is enhanced in papillary thyroid cancer [10,27,28]. Studies have shown direct correlation
between the expression of MMPs and tumor invasion
and metastasis [29]. Aggressive variants of thyroid cancers tend to express
more MMP-2 [10]. Thyroid cancer cell
lines - BCPAP (poorly differentiated thyroid cancer), K1 (papillary thyroid cancer), CGTH-W-1 (follicular
thyroid cancer with metastasis to sternum) and FTC133 (follicular thyroid cancer) also express MMP-2 [19,30].
Anaplastic thyroid cancer cell line SW 579
also express MMP2 [31]. Increased expression of MMP-2 is also seen in
urothelial, prostate, breast,
stomach cancers and in gliomas. Chlorotoxin has been shown to help in the
endocytosis of MMP-2 [7]. Study by
Kalhori and Törnquist demonstrated that MMP-2 is involved in ML-1 cell line’s invasive potential [32].
In our study, unconjugated
chlorotoxin did not show any significant reduction in cancer cell viability when compared to untreated controls (Figure 1B). It was hypothesized that a Chlorotoxin and Saporin conjugate would
inhibit the cell growth of ML-1 thyroid cancer
cells. We observed a significant dose-dependent inhibition of ML-1 cell
viability when treated with 40-600
nm of conjugated toxin (Figure 2A).
The most statistically significant reduction of ML-1 thyroid cancer cell viability occurred with treatment of
600 nm of CTX-SAP. Cell viability was
decreased by 49.77% with 600 nm of CTX-SAP when compared with non-treated
controls (Figure 2B).
Further studies are needed before this can be used in clinical
practice. This study needs to be
replicated in other thyroid cancer cell lines also. Reliable methods to detect
the presence of MMP-2 in cancer cell
lines will help us identify potential malignancies that can be treated with CTX-SAP. Radioiodine[131/125I] labelled synthetic
Chlorotoxin has been successfully
administered without any significant side effects in vivo [33-36]. This could be used to image tumors that are RAI-resistant but express MMP2. Further
studies are also needed to understand the effect of this toxin
on tumor microenvironment and surrounding normal
cells.
CONCLUSION
Our
study demonstrated that CTX-SAP decreased cell viability of ML-1 cells which express MMP-2. This study
points towards MMP-2 as a potential target for
RAI-resistant thyroid cancer. Further studies are needed to
develop safe and effective treatment against
aggressive thyroid cancer using CTX-SAP.
ACKNOWLEDGMENT
We thank the Department of Biology at Missouri State University,
Springfield, Missouri, United
States for facilitating this study. Additionally, we are indebted to Hazzar Abysalamah, Hanna Williams, and
Dr. Christopher Lupfer for help with cell culture.
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