Autophagy: A new target for advanced papillary thyroid cancer therapy

doi:10.1016/j.surg.2009.09.019

Surgery

Volume 146, Issue 6, December 2009, Pages 1208-1214

https://doi.org/10.1016/j.surg.2009.09.019 Get rights and content

Background

Autophagy is a conserved response to stress that facilitates cell survival in some contexts and promotes cell death in others. We sought to characterize autophagy in papillary thyroid cancer (PTC), and to determine the effects of autophagy inhibition on chemosensitivity and radiosensitivity.

Methods

The human thyroid papillary carcinoma cell lines TPC-1 and 8505-C were treated with doxorubicin or radiation in the presence or absence of the autophagy-specific inhibitor 3-methyladenine (3-MA).

Results

Although light chain 3 (LC3)-II protein levels were undetectable in normal thyroid and PTC specimens at baseline, doxorubicin exposure induced LC3-II expression and the formation of autophagosomes. Both PTC cell lines expressed low levels of LC3-II under standard conditions. Treatment of these cells with doxorubicin strongly induced LC3-II expression and the formation of autophagosomes; however, doxorubicin–mediated induction of LC3-II was abrogated by 3-MA. Moreover, 3-MA significantly increased the doxorubicin IC₅₀ in both PTC cell lines. Radiation exposure also induced LC3-II expression. Treatment with 3-MA abrogated the radiation–induced increase in LC3-II in both cell lines and reduced radiosensitivity by 49% and 31% in 8505-C and TPC-1 cells, respectively.

Conclusion

Autophagy inhibition promotes PTC resistance to doxorubicin and radiation. Therefore, autophagy activation may be a useful adjunct treatment for patients with PTC that is refractory to conventional therapy.

Section snippets

Materials

Microtubule-associated protein 1 light chain 3 (LC3) immunoblotting is a technique used widely for the detection of autophagic activity.¹⁴ LC3-II protein is incorporated into autophagosomes, and the protein expression level serves as a good indicator of autophagic activation.¹⁵ Mouse anti-human LC3 antibody was purchased from MBL International (Nagoya, Japan). Mouse anti-actin antibody pan Ab-5 was purchased from Neomarker (Fremont, CA). Secondary horseradish peroxidase-conjugated anti-mouse

Doxorubicin induces autophagy in freshly harvested human PTC specimens

By immunoblot analysis, LC3-I and -II protein expression was undetectable in freshly harvested thyroid and PTC specimens (Fig 1, A); however, exposure of PTC to doxorubicin for 8 hours induced the expression of LC3-II protein ex vivo (Fig 1, B). Furthermore, PTC exposed to doxorubicin ex vivo induced the formation of autophagosomes, which was evident on electron microscopy (Fig 1, C). This observation suggests that autophagic activity is low at baseline in PTC specimens from patients at the

Discussion

Autophagy is an evolutionarily conserved process that entails the degradation and recycling of organelles during times of cellular stress.²² In theory, autophagy could enable cancer cell survival during times of cellular stress from chemotherapy exposure, ionizing radiation, or hypoxia. Thus, autophagy could protect cancer cells from unfavorable tumor microenvironments and promote resistance to cancer therapies.

Autophagy, however, may also promote cancer cell death. In cells that lack the

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Curcumin, a polyphenolic compound, is a well-known anticancer agent, although its poor bioavailability remains a big concern. Recent studies suggest that autophagy-targeted therapy may be a useful adjunct treatment for patients with thyroid cancer. Curcumin acts as an autophagy inducer on many cancer cells. However, little is known about the exact role of curcumin on thyroid cancer cells. In the present study, curcumin significantly inhibited the growth of thyroid cancer cells. Autophagy was markedly induced by curcumin treatment as evidenced by an increase in LC3-II conversion, beclin-1 accumulation, p62 degradation as well as the increased formation of acidic vesicular organelles (AVOs). 3-MA, an autophagy inhibitor, partially rescued thyroid cancer cells from curcumin-induced cell death. Additionally, curcumin was found to exert selective cytotoxicity on thyroid cancer cells but not normal epithelial cells and acted as an autophagy inducer through activation of MAPK while inhibition of mTOR pathways. Hyperactivation of the AKT/mTOR axis was observed in the majority of PTC samples we tested, and thyroid cancer cell lines along with cancer tissue specimens sustained a low basal autophagic activity. Taken together, our results provide new evidence that inducing autophagic cell death may serve as a potential anti-cancer strategy to handle thyroid cancer.
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Therefore, we believe that BCPAP cells protect themselves against the damage and apoptosis caused by high iodine using autophagy. It was found that autophagy could be activated rapidly when PTC tissues were exposed to harmful factors such as chemotherapeutic drugs and external radiation, which further confirmed our conclusions [28]. Iodine is a suitable stimulant for BCPAP cells, which does not cause stress and only develops autophagy at low levels.
Papillary thyroid carcinoma (PTC) is a common endocrine tumor. This study found that different iodine concentrations affected the proliferation, apoptosis, and migration of PTC. For this study, we collected clinical information from PTC patients and monitored the levels of urinary iodine, LC3-II, and caspase-3 in cancer tissue, and BRAF kinase in peripheral blood from PTC patients. We also monitored the proliferation, apoptosis and migration ability of human papillary-thyroid carcinoma (BCPAP) cells at different iodine concentrations and their association with changes in autophagy and BRAF kinase activity of BCPAP cells at high iodine levels (10⁻³ mol/l). We found that the proportion of tumor diameters ≥ 1 cm in the iodine excess group were lower than that in the iodine non-excess group. The proportion of PTC patients with infiltration in the iodine excess group was higher than that in the iodine non-excess group. Levels of the autophagy-related protein LC3-II and the apoptosis-related protein caspase-3 in cancer tissues, and activity of BRAF kinase in peripheral blood, were positively correlated with urinary iodine concentrations from PTC patients. At high iodine levels, the proliferation rate decreased, and apoptosis percentage and migration rates increased compared with the no-iodine group. At high iodine levels, the frequencies of autophagosomes (Aph) and autophagosome-lysosomes (Apl) in BCPAP cells increased significantly, and activities of LC3-II and BRAF kinase increased, respectively. The activity of LC3-II decreased when BRAF kinase was inhibited. The activity of LC3-II and the proliferation and migration rates of BCPAP cells decreased, and the apoptosis percentage increased when autophagy was inhibited at high iodine concentrations. Our results demonstrated that, in the presence of excessive iodine, the mean tumor size of PTC patients was smaller and easier to invade than tumors in patients not supplied with excessive iodine. The levels of autophagy and apoptosis in PTC cancer tissues, and activities of BRAF kinase in peripheral blood increased with increasing urinary iodine concentrations. High iodine levels inhibited cell proliferation and promoted apoptosis and migration of PTC cells. Autophagy induced by BRAF kinase in PTC cells was involved in anti-apoptosis, and promoted proliferation and migration at high iodine concentrations. This study provides a rationale for iodine supplementation in PTC patients.
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Furthermore, researchers have also begun to focus on the autophagy pathway with respect to the pathogenesis and development of thyroid cancer, particularly in tumors that are not sensitive to conventional therapy. Autophagy responds quickly to myriad internal and external environmental pressures that exist in eukaryotic cells and that happen during the aging process [28,29]. Autophagy not only is a cell survival mechanism but also can induce cell death.
Dietary anthocyanin compounds have multiple biological effects, including antioxidant, anti-inflammatory, and anti-atherosclerotic characteristics. The present study evaluated the anti-tumor capacity of mulberry anthocyanins (MA) in thyroid cancer cells. Our data showed that MA suppressed SW1736 and HTh-7 cell proliferation in a time- and dose-dependent manner. Meanwhile, flow cytometry results indicated that MA significantly increased SW1736 and HTh-7 cell apoptosis. We additionally observed that SW1736 and HTh-7 cell autophagy was markedly enhanced after MA treatment. Importantly, anthocyanin-induced cell death was largely abolished by 3-methyladenine (3-MA) or chloroquine diphosphate salt (CQ) treatment, suggesting that MA-induced SW1736 and HTh-7 cell death was partially dependent on autophagy. In addition, activation of protein kinase B (Akt), mammalian target of rapamycin (mTOR), and ribosomal protein S6 (S6) were significantly suppressed by anthocyanin exposure. In summary, MA may serve as an adjunctive therapy for thyroid cancer patients through induction of apoptosis and autophagy-dependent cell death.
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Autophagy protects cancer cells from cell death via catabolic processes during nutrient shortage or exposure to chemotherapeutic agents [13,14]. Alternatively, autophagy can induce cancer cell death by destroying essential cellular components, tipping cells into apoptosis [15–17]. There is a balance inherent in the autophagic process that can either promote or inhibit cancer cell growth and proliferation.
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: Supported by research grants from the Department of Surgery, Brigham and Women's Hospital, Boston, MA.

: Presented at the 30th Annual Meeting of the American Association of Endocrine Surgeons, Madison, Wisconsin, May 2–5, 2009.

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American Association of Endocrine SurgeonAutophagy: A new target for advanced papillary thyroid cancer therapy

Background

Methods

Results

Conclusion

Section snippets

Materials

Doxorubicin induces autophagy in freshly harvested human PTC specimens

Discussion

Am J Med

Int J Biochem Cell Biol

Curr Opin Cell Biol

Biochem Biophys Res Commun

Cell

Cell

J Mol Biol

J Biol Chem

Identification of genes differentially expressed in benign versus malignant thyroid tumors

Clin Cancer Res

Chemotherapy with doxorubicin in progressive medullary and thyroid carcinoma of the follicular epithelium

Horm Metab Res

Autophagy

Ann N Y Acad Sci

Autophagy as a regulated pathway of cellular degradation

Science

Why sick cells produce tumors: the protective role of autophagy

Autophagy

A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles

Cancer Res

Autophagy: a novel mechanism of synergistic cytotoxicity between doxorubicin and roscovitine in a sarcoma model

Cancer Res

American Association of Endocrine Surgeon
Autophagy: A new target for advanced papillary thyroid cancer therapy