Research ArticleCelastrol induces unfolded protein response-dependent cell death in head and neck cancer
Introduction
A remarkable incidence of oral squamous cell carcinoma (OSCC) persists in the United States. Progress in durable patient responses has been only incremental since the introduction of cisplatin in 1978, and approximately 40,000 Americans will be newly diagnosed with oral cavity and oropharyngeal cancer this year [2]. In an attempt to improve the outcomes of conventional chemotherapy we have utilized a cell-based high throughput screening technique to identify novel small molecules that intensify the unfolded protein response (UPR) and selectively kill malignant cells [1].
The unfolded protein response (UPR) is a cell׳s rejoinder to the accumulation of misfolded protein in the lumen of the endoplasmic reticulum (ER). Innositol-requiring enzyme 1 alpha (IRE1α), activating transcription factor 6 (ATF6), and protein kinase RNA-like endoplasmic reticulum kinase (PERK) are three ER transmembrane sensors that continuously monitor the status of luminal protein folding. Broadly, the UPR consists of genetically distinct pathways that are simultaneously activated to either adapt to a folding challenge or initiate cell death when a stress is particularly robust or protracted. The adaptive response is directed primarily through the un-conventional splicing of a 26 base intron from X-box binding protein (XBP1) mRNA by IRE1αSpliced XBP1 yields a potent transcription factor that increases the expression of foldases, chaperones and other heat shock factors and glucose-related proteins that return to the ER in an attempt to remedy the folding defect. Although the precise mechanism governing the switch from UPR-mediated adaptation toward cell death remains an incomplete story, it is clear, when the adaptive response becomes overwhelmed, that the activation of the PERK-eIF2α-axis induces ATF4 and CHOP expression prior to apoptosis [3].
Given the rapid growth rate and highly secretory nature of many solid and hematological tumors it is not surprising that many human cancers are characterized by increased expression of translation factors and high basal levels of UPR signaling and stress. Recent studies have revealed increased expression of eukaryotic initiation factors (eIFs) and UPR-related chaperones in breast [4], [5], bladder [6], lung [7], thyroid [8], lymphoma [9], colorectal [10], leukemia [11], larynx [12], [13] and OSCC [11], [14], [15]. As malignant cell populations begin to grow and invade host tissue the extracellular tumor milieu becomes increasingly starved of oxygen, glucose and other nutrients as the rate of expansion outpaces the capacity of its vasculature. The cellular stress caused by these harsh conditions leads to IRE1α- and PERK (ATF4) -mediated angiogenesis and cell survival [16]. Importantly, Xbp1-/- fibroblasts and Xbp1 knockdown cells failed to form tumors in mice [17], and PERK -/- cells and xenografts are unable to tolerate hypoxia [18]. When considered together, the increased expression of translation factors and the demand for UPR-driven angiogenesis in the tumor stroma strongly support the hypothesis that targeting the UPR with small molecules will be a productive therapeutic approach.
A cell-based high throughput screen was engineered using CHO cells transfected with luciferase reporters to specifically monitor XBP1 splicing or CHOP promoter activation [1]. A library of approximately 66,000 compounds was screened at the University of Michigan Center for Chemical Genomics and celastrol, a triterpenoid compound isolated from the plant family Celastraceae [19], emerged as a hit that could activate both UPR reporters.
Although most studies have focused on the anti-inflammatory properties of celastrol, there are a growing number of reports highlighting its use as a promising anti-cancer compound in breast leukemia, melanoma, myeloma, pancreatic and prostate cell culture and xenograft models [20], [21], [22], [23], [24]. The anti-proliferative effects of celastrol have been attributed to mechanisms involving diverse signaling networks that include the inhibition of pro-survival NF-B signaling, proteasome inhibition and the up-regulation of pro-apoptotic Bcl-2 family members and down regulation of anti-apoptotic genes such as Bcl-2 and XIAP. Since celastrol has been shown to potently induce the expression of heat shock proteins [25] and was able to activate UPR luciferase reporters in our screen, we hypothesized that its ability to induce cell death and reduce xenograft tumor burden in several models might be dependent on its ability to activate the UPR.
Section snippets
Cell lines, reporters and reagents
Stably transfected CHO-K1 cells containing pathway-specific luciferase reporters for the PERK/eIF2α/CHOP pathway or the IRE1/XBP1 pathway were used for screening as described [1]. The human floor of mouth squamous cell carcinoma lines UMSCC1, UMSCC14A and laryngeal squamous cell carcinoma cell line UMSCC23 were kindly provided by Dr. Thomas Carey at the University of Michigan. The tongue carcinoma cell line CAL27 (CRL-2095), the salivary epidermoid carcinoma cell line A-253 (HTB-41) and the
Celastrol activates the unfolded protein response
The tripterine celastrol emerged as a hit in a high throughput screen to identify small molecule activators of the unfolded protein response (UPR). Confirmatory dose-response assays demonstrated activation of the CHOP- and XBP1-luciferase reporters used in the primary screen [1], [28] (Fig. 1A). The closely related analog dihydrocelastrol was active only at substantially higher concentrations (data not shown). A panel of oral squamous cell carcinoma (OSCC) cell lines exposed to celastrol
Discussion
The recent surge of publications delineating molecular mechanisms by which natural products act as anticancer agents is reflective of a world-wide desire to identify novel cost-effective therapies from the biota. Many natural products including celastrol have been used in traditional culture-specific medical practices for hundreds of years and offer tremendous promise in that their ability to be administered and tolerated in humans is largely known. Celastrol is derived from Tripteryguim
Acknowledgments
This research was supported by DE019678, the Wayne State University Fund for Medical Research and the Children׳s Research Foundation of Michigan (A.M.F.) and DK088227, HL057346, DK0422394 (R.J.K).
References (50)
- et al.
Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response
J. Biomol. Screen.
(2011) - et al.
Expression of the eukaryotic translation initiation factors 4E and 2alpha in non-Hodgkin׳s lymphomas
Am J. Pathol.
(1999) - et al.
Molecular targeting of the oncogene eIF4E in acute myeloid leukemia (AML): a proof-of-principle clinical trial with ribavirin
Blood
(2009) - et al.
Celastrol isolated from Tripterygium regelii induces apoptosis through both caspase-dependent and -independent pathways in human breast cancer cells
Food Chem Toxicol: Int J. published Br.Indus. Biol. Res. Assoc.
(2011) - et al.
Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance
J. BiolChem.
(1997) - et al.
The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status
Blood
(2006) - et al.
Celastrol, a novel triterpene, potentiates TNF-induced apoptosis and suppresses invasion of tumor cells by inhibiting NF-kappaB-regulated gene products and TAK1-mediated NF-kappaB activation
Blood
(2007) - et al.
Translational control is required for the unfolded protein response and in vivo glucose homeostasis
Mol. Cell
(2001) - et al.
Herbal medications commonly used in the practice of rheumatology: mechanisms of action, efficacy, and side effects
Sem. Arthr. Rheum.
(2005) - et al.
Celastrols as inducers of the heat shock response and cytoprotection
J. Biol. Chem.
(2004)
Chemical and biological approaches synergize to ameliorate protein-folding diseases
Cell
Celastrol-induced apoptosis in human HaCaT keratinocytes involves the inhibition of NF-kappaB activity
EurJ. pharmacol.
Celastrol inhibits the growth of human glioma xenografts in nude mice through suppressing VEGFR expression
Cancer Lett.
Cancer statistics, 2009
CA: Cancer J. Clin.
ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death
Nat. Cell Biol.
Eukaryotic translation initiation factor 4E induced progression of primary human mammary epithelial cells along the cancer pathway is associated with targeted translational deregulation of oncogenic drivers and inhibitors
Cancer Res.
The proto-oncogene/translation factor eIF4E: a survey of its expression in breast carcinomas
Int. J. Cancer
Eukaryotic initiation factor-4E in superficial and muscle invasive bladder cancer and its correlation with vascular endothelial growth factor expression and tumour progression
Br J Cancer
Expression of eukaryotic translation initiation factors 4E and 2alpha is increased frequently in bronchioloalveolar but not in squamous cell carcinomas of the lung
Cancer
Expression of eukaryotic translation initiation factors 4E and 2alpha correlates with the progression of thyroid carcinoma
Thyroid
Upregulation of protein synthesis initiation factor eIF-4E is an early event during colon carcinogenesis
Oncogene
Detection of the proto-oncogene eIF4E in larynx and hypopharynx cancers
Arch. Otolaryngol. Head Neck Surg.
Autophagy provides nutrients but can lead to Chop-dependent induction of Bim to sensitize growth factor-deprived cells to apoptosis
Mol. Biol. Cell
Detection of the proto-oncogene eIF4E in surgical margins may predict recurrence in head and neck cancer
Oncogene
The NF-kappa B inhibitor, celastrol, could enhance the anti-cancer effect of gambogic acid on oral squamous cell carcinoma
BMC Cancer
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