Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/β-catenin and Sonic

Hedgehog Pathways
Jian-Yun Zhu, Xue Yang, Yue Chen, Ye Jiang, Shi-Jia Wang, Yuan Li, Xiao-Qian Wang,
Yu Meng, Ming-Ming Zhu, Xiao Ma, Cong Huang, Rui Wu, Chun-Feng Xie, Xiao-Ting Li,
Shan-Shan Geng, Jie-Shu Wu, Cai-Yun Zhong and Hong-Yu Han

Abstract
Cancer stem cells (CSCs) are highly implicated in the progression of human cancers. Thus,
targeting CSCs may be a promising strategy for cancer therapy. Wnt/β-catenin and Sonic Hedgehog
pathways play an important regulatory role in maintaining CSC characteristics. Natural compounds,
such as curcumin, possess chemopreventive properties. However, the interventional effect of curcumin
on lung CSCs has not been clarified. In this study, tumorsphere formation assay was used to enrich lung
CSCs from A549 and H1299 cells. Levels of lung CSC markers (CD133, CD44, ALDHA1, Nanog and
Oct4) and the number of CD133-positive cells were significantly elevated in the sphere-forming cells.
Curcumin also efficiently eliminated lung CSC traits, as evidenced by reduced tumorsphere formation,
reduced number of CD133-positive cells, decreased expression levels of lung CSC markers, as well as
proliferation inhibition and apoptosis induction. Moreover, curcumin suppressed the activation of both
Wnt/β-catenin and Sonic Hedgehog pathways. In conclusion, the results suggested that curcumin
exhibited its interventional effect on lung CSCs via inhibition of Wnt/β-catenin and Sonic Hedgehog
pathways. These novel findings could provide new insights into the potential therapeutic application of
curcumin in lung CSC elimination and cancer intervention.

Introduction
The growth of tissues and reproduction of the cells in our bodies are regulated by key
sets of DNA instructions. However, these DNA sequences can be disrupted – through the action
of viruses, environmental factors such as radiation or toxins, mutations transcription errors or
inborn genetic flaws, which can lead to the faulty regulation of the cells. These changes will
ultimately result to the abnormal growth of the self-protective and opportunistic cancer cells.
Lung Cancer is one of the deadliest malignant tumors in the world. The survival rate of
those diagnosed with lung cancer remains low despite advancements in the cancer treatment.
Recently, researchers have developed a new cancer treatment based on the eradication of
cancer stem cells. Cancer stem cells (CSCs) are defined as a rare subset of tumor cells with the
ability of continuous self-renewal and differentiation. Important properties of CSCs include
self-renewal through asymmetrical and symmetrical division, ability to spread cancer in other
areas in the body or Metastasis, and resistance to conventional therapy as it can initiate self-
renewal. CSCs are critically implicated in the initiation, progression, drug resistance and
recurrence of human cancers. Thus, targeting CSCs are important in cancer treatments.
There are several pathways that initiate the occurrence of CSCs, among these are
Wnt/B-catenin and Sonic Hedgehog (SHh) pathways. Wnt/B-catenin pathway is characterized
by the expression level of the multifunctional protein B-catenin, a key mediator in this pathway.
B-catenin is regulated by the destruction complex composed of glycogen synthase kinase 3β
(GSK3β), casein kinase 1α, Axin and adenomatous polyposis coli (APC). Upon the absence of
the Wnt signaling, the cytoplasmic B-catenin will be degraded by the destruction complex and
upon the activation of the Wnt signaling, B-catenin translocates into the nucleus where it
activates the transcription of downstream genes including c-Myc, Cyclin D1, CD44 and
ALDH. On the other hand, Sonic hedgehog pathway (Hh) is initiated by the binding of the Hh
ligand to its receptor Patched (PTCH). When the Hh ligand is absent, the PTCH represses the
signal transduction by inhibiting the activity of the transmembrane protein Smoothened (Smo).
The activation of Hh binding, on the contrary, results in the activation of Gli transcription
factors family including Gli1 and Gli2. Genes from the Wnt/B-catenin and Sonic hedgehog
pathway (c-Myc, Cyclin D1, CD44, ALDH, Gli1 and Gli2) are target genes that promote
several oncogenic properties including cancer stem cells or CSCs.
In this study, curcumin, a polyphenol isolated from the rhizomes of turmeric (Curcuma
longa) was tested against lung cancer stem cells. Curcumin’s anticancer properties have been
exhibited in hepatocellular and esophageal carcinoma cell lines. However, its effect on the
Wnt/B-catenin and Sonic hedgehog pathways remains unclear. Thus, this study aims to
investigate the inhibitory effects of curcumin on the characteristics of lung CSCs as well as its
activity against Wnt/B-catenin and Hh pathways.
Methodology
The general methodology of the study is broken into to four parts: (1) Tumorsphere
formation assay, (2) Treatment of sphere-forming cells with and its activity against lung CSCs,
(3) Assessment of the treatment of curcumin in the cell proliferation and apoptosis of lung
CSCs, and (4) Assessment of the treatment of curcumin in the suppression of Wnt/B-catenin
and Shh pathways in lung CSCs.
Tumorsphere formation assay
A549 and H1299 cells were cultured in serum-free medium with epidermal growth
factors. The cells were then plated into a 24-well plate, and fresh medium was added every two
days. Tumorsphere formation was observed and photographed by a light microscope.
To verify the plated lung CSCs, the protein expression levels of lung CSC markers
(CD133, CD44, ALDH1A1, Nanog and Oct4) were measured by western blot analysis.
Western blot analysis is used to separate and identify proteins. In this technique, a mixture of
proteins is separated based on molecular weight, and through gel electrophoresis. The results
are transferred to a membrane, embedded with antibodies specific to the protein of interest,
producing a band for each protein. In this study, cells were collected, washed with ice-cold
phosphate buffer saline (PBS) and the solubilized in radioimmunoprecipitation assay lysis
buffer containing protease inhibitors. Afterwards, the supernatant was used for western blot
analysis. Protein concentrations were determined by bicinchoninic acid protein assay and were
standardized among the samples. Proteins were subjected to sodium dodecyl sulfate
polyacrylamide gel electrophoresis and were transferred to the polvvynylidene difluoride
membranes. The membranes were then incubated with the appropriate primary antibodies.
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control to
measure the expression of each protein.
To further characterize the lung CSCs, the number of CD133 positive cells were
measured by flow cytometry. Flow cytometry is a technique that measures optical and
fluorescence characteristics of single cells. Fluorescent dyes bind or intercalates with different
components such as DNA or RNA. In addition, antibodies conjugate to fluorescent dyes can
bind specific proteins on cell membranes or inside cells. When labelled cells are passed by a
light source, the fluorescent molecules are excited to a higher energy state and retunes back to
their resting states, emitting light energy at higher wavelengths. This allows several cell
properties to be measured simultaneously. In this study, cells were centrifuged and washed
with cold PBS. Cells (1 × 106) were incubated with APC-conjugated human monoclonal
CD133/1 antibody, them measured by flow cytometry.
Treatment of sphere-forming cells with curcumin and its activity against lung CSCs
To analyze the inhibitory function of curcumin on lung CSCs, A549 and H1299 sphere-
forming cells were treated with various concentrations of curcumin (0, 5, 10, 20 and 40 μM),
using 0.1% dimethyl sulfoxide as control. Seven days after treatment, the tumorspheres of each
group were imaged. Western blotting was performed to analyze the expression levels of lung
CSC markers and also, flow cytometry to detect CD133-positive cells in 1549 sphere-forming
cells.
Assessment of the treatment of curcumin in the activity of lung CSCs
A549 and H1299 sphere-forming cells were treated with appropriate concentrations of
curcumin for 7 days. Western blot analysis was performed to detect the expression levels of
proliferation proteins (PCNA and cyclin D1) and apoptosis proteins (Bcl2, Bax, Cleaved
Caspase 8, Cleaved Caspase 9 and Cleaved Caspase 3). Lastly, flow cytometry was conducted
to detect apoptotic cells in A549 sphere-forming cells.
Assessment of the treatment of curcumin in the suppression of Wnt/B-catenin and Shh pathways
in lung CSCs
A549 and H1299 sphere-forming cells were incubated with various concentrations of
curcumin for 7 days. The protein expression levels of Wnt/β-catenin pathway (GSK3β, p-
GSK3β, β-catenin and c-Myc) were determined using western blotting. Likewise, protein
expression levels of Sonic Hedgehog pathway (shh, Smo, Gli1 and Gli2) were detected by
western blotting analysis.
Results and Discussion
The results and discussion of the study are outlined as follows:
1. Tumorsphere formation of lung cancer cells with SFM culture in vitro
Cancer stem cells are characterized by their tumorrsphere-forming ability in in-vitro.
Tumorsphere formation assay via Serum free medium (SFM) culturing is used in the
study. As shown in Figure 1A, both A549 and H1299 cells formed stable tumorspheres
in the SMF culture. The expression of several distinct lung CSC makers such as CD133,
CD44, ALDH1A1, Nanog and Oct4 were analyzed through western blot analysis. As
shown in Figure 1B, both the cells expressed elevated protein levels of the CSC
markers. In addition, the percentage of the CD133-positive cells was increased in the
 sphere forming cells as shown in Figure 1C. These results suggest the characteristics of
CSCs in the sphere-forming lung cancer cells.

Figure 1. Tumorsphere formation of lung

cancer stem cells (CSCs) by serum-free
medium (SFM) culture: (A) Representative
pictures were imaged under a light microscope.
(B) The protein expression levels of lung CSC
markers (C) Detection of CD133-positive cells
in A549 cells.

2. Curcumin diminishes the activity of lung CSCs

The efficacy of cucumin against CSCs have been reported on several human cancers.
In this study, the effect of curcumin against lung CSCs were explored. Results showed
that curcumin inhibited the formation of both A549 and H1299 tumorspheres in a
concentration-dependent manner (Figure 2A). It was also noted that the curcumin was
more potent against H1299 tumorspheres. In addition, mRNA and protein expression
levels of lung CSC markers were decreased by curcumin (Figure 2D). Flow cytometry
showed that curcumin significantly reduced the number of CD133-positive cells in
A549 tumorpsheres (Figure 2E and F).
 Figure 2. Curcumin eliminates the traits of lung CSCs. (A) The representative images of A549
and H1299 sphere-forming cells (B) Quantitative real-time PCR (C) and western blotting (D)
were performed to analyze the expression levels of lung CSC markers (CD133, CD44,
ALDH1A1, Nanog and Oct4). (E and F) Detection of CD133-positive cells in A549 sphere-
forming cells by flow cytometry assay.

3. Curcumin inhibits cell proliferation and induces apoptosis of lung CSCs

Effects of curcumin on cell proliferation and apoptosis was investigated. As shown in

Figure 3A, the levels of cell proliferation-related proteins PCNA and Cyclin D1 were
reduced in both A5449 and H1299 sphere-forming cells. Also, curcumin treatment
resulted in a decrease of level of anti-apoptosis protein Bcl2, whereas the levels of pro-
apoptosis proteins Bax and caspases were increased. Lastly, flow cytometry analysis
 also indicated that curcumin-induced apoptosis in A549 sphere-forming cells. All in all,
the results showed that curcumin inhibited proliferation and induced apoptosis of lung
CSCs.

Figure 3. Curcumin inhibits proliferation and

induces apoptosis in lung CSCs. (A) Western blot
assay for the detection of the expression levels of
proliferation proteins (PCNA and cyclin D1). (B)
Western blot assay for the detection of the
expression levels of apoptosis proteins (Bcl2, Bax,
Cleaved Caspase 8, Cleaved Caspase 9 and
Cleaved Caspase 3). (D) Flow cytometry detection
of apoptotic cells in A549 sphere-forming cells

4. Curcumin suppresses Wnt/β-catenin pathway in lung CSCs

Wnt/β -catenin plays a crucial role in the maintenance of CSCs. Activation of

this pathway depends upon the key regulator β-catenin which is regulated by the
destruction complex. In this study, the treatment of curcumin was assessed if it could
downregulate the Wnt/β -catenin pathway in the lung CSCs. As shown in Figure 4A,
curcumin treatment decreased the level of p-GSK3β (Ser9) and β-catenin, as well as its
downstream target genes (c-Myc and Cyclin D1). It was also shown to increase the
GSK3β. All in all, the results indicated the inhibition of Wnt/β-catenin pathway in lung
CSCs by curcumin (Figure 4A).

5. Curcumin downregulates Sonic Hedgehog pathway in lung CSCs

Like the Wnt/β-catenin pathway, Sonic Hh pathway also plays a critical role in
regulating CSC activity. When Shh ligand binds to PTCH receptor, PTCH relieves its
inhibition on Smo and leads to activation of Gli transcription factors family. In this
study, it was showed that the Sonic Hh pathway components, including shh, Smo, Gli1
and Gli2, were markedly downregulated by curcumin in A549 and H1299 sphere-
forming cells in a concentration-dependent manner, indicating the suppression of this
pathway (Figure 5A).

Figure 4. Curcumin suppresses lung CSCs via Wnt/β-catenin inhibition. (A) The
protein expression levels of Wnt/β-catenin pathway (GSK3β, p-GSK3β, β-catenin and
c-Myc) determined using western blotting.

Figure 5. Curcumin represses lung CSCs via Sonic Hedgehog inhibition. (A) A549 and
H1299 sphere-forming cells were treated with different concentrations of curcumin for
7 days. The protein expression levels of Sonic Hedgehog pathway (shh, Smo, Gli1 and
Gli2) detected by western blotting.
Conclusion

The study revealed that curcumin significantly inhibited lung CSC activities, as
manifested by suppression of tumosphere formation, lung CSC marker expression and
proliferation, as well as apoptosis induction. It was also illustrated that downregulation
of Wnt/β-catenin and Sonic Hh pathways mediated the inhibitory effects of curcumin
on lung CSCs. Findings from this study could provide new insights into the molecular
mechanisms and the potential therapeutic application of curcumin in lung CSC
elimination as well as cancer intervention.

Recommendations

Specificity of curcumin as a potential anti-cancer stem cell drug must be

assessed. As a potential anti-cancer drug, effects to the healthy neighboring non-cancer
cells must be minimum to prevent further toxicity in the body. Also, in-vivo models
and clinical trials must be performed.

References

Reya, & Clevers. (2005). Wnt signalling in stem cells and cancer. Nature, 434(7035),
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dopt=AbstractPlus&list_uids=15829953\npapers://01628a62-bdc0-47f5-904e-
9689163247b2/Paper/p341

Zhu, J.-Y., Yang, X., Chen, Y., Jiang, Y., Wang, S.-J., Li, Y., … Han, H.-Y. (2017).
Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/β-catenin
and Sonic Hedgehog Pathways. Phytotherapy Research : PTR, (October
2016). https://doi.org/10.1002/ptr.5791