Cordycepin Induces Apoptosis through JNK-Mediated Caspase Activation in Human OEC-M1 Oral Cancer Cells

Cordycepin, a bioactive compound extracted from Cordyceps sinensis, can induce apoptosis in human OEC-M1 oral cancer cells. However, the exact mechanism is still unclear. The present study aimed to investigate the underlying mechanism of cordycepin-induced apoptosis in OEC-M1 cells. Following treatment with cordycepin, apoptosis was examined and quantified using a DNA laddering assay and a cytokeratin 18 fragment enzyme-linked immunosorbent assay, respectively. Expressions of mitogen-activated protein kinases (MAPKs) and apoptosis-related proteins were detected by the western blot analysis. Our results show that a pan-caspase inhibitor, Z-VAD-FMK, could significantly inhibit cordycepin-induced apoptosis in OEC-M1 cells. In addition, treatment with cordycepin not only activated caspase-8, caspase-9, and caspase-3 but also induced Bid and poly ADP-ribose polymerase cleavages. Furthermore, cordycepin also induced the activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase, and p38 MAPKs. Among MAPKs, activation of JNK solely contributed to cordycepin-induced apoptosis with the activation of caspase-8, caspase-9, and caspase-3 and cleavage of PARP. Taken together, the present study demonstrated that cordycepin activated JNK and caspase pathways to induce apoptosis in OEC-M1 cells.


Introduction
Oral cancer is a subgroup of squamous cell carcinoma of the head and neck, presenting on the lips, mouth, and upper throat around the oral cavity.High-risk factors for developing oral cancers include tobacco use, betel quid chewing, alcohol consumption, and oral chronic infammation [1,2].With a high incidence, oral cancer has been a public health problem in India, Sri Lanka, Tailand, South China, and Taiwan [3].Although advances have been made in clinical treatments, the 5-year survival rate of oral cancer patients after surgery and radiotherapy treatments remains low, at about 50%, and has not been improving in the past few decades [4].Tus, there is an urgent and large-scale need for novel, efective drugs to improve the prognosis of oral cancer patients.
Recent studies have shown that extracts from certain plants in the wild have high application value in antiobesity and diabetes therapy, neuromodulation, antivirus activity, and nanomaterial synthesis [5][6][7][8][9].Plant extracts have also been used to fght cancers.For instance, certain small molecules from natural sources have been used to treat breast cancer [10].Another example is curcumin, a natural bioactive compound extracted from Curcuma longa, which was shown to induce apoptosis of cancer cells and suppress tumor growth [11][12][13].Yet another, paclitaxel, a substance extracted from the Pacifc yew tree, Taxus brevifolia, is clinically used to treat various cancers [14].
In this study, we have focused on cordycepin, a bioactive compound extracted from Cordyceps sinensis, which has multiple documented pharmacological activities in human and animal models, including immunomodulatory [15], anti-infammatory [16], and hypoglycemic efects [17].Furthermore, several studies have demonstrated that cordycepin not only has the ability to induce apoptosis in various cancer cell lines in vitro [18][19][20] but also suppresses tumor growth in vivo [18,19], suggesting that the antitumor efects of cordycepin are related to apoptosis.
We reported earlier that cordycepin could induce apoptosis in human OEC-M1 oral cancer cells [29].However, the mechanism involved in the cordycepin-induced apoptotic efects remains to be elucidated.Here we investigated those mechanisms in OEC-M1 oral cancer cells because the insights thus gained have great therapeutic potential for oral cancer.Te results of these experiments revealed that cordycepin activated JNK and caspase pathways to induce apoptosis in OEC-M1 cells.Our fndings can be used to design more successful therapy regimes combined with cordycepin anticancer efects to improve the poor prognosis of patients with oral cancers.

Enzyme-Linked Immunosorbent Assay (ELISA).
Cytokeratin 18 fragment levels in the cell culture supernatants were quantifed by using the SimpleStep ELISA kits (Cat.No. ab254515; Abcam, Plc., Cambridge, UK) according to the manufacturer's instructions.

Western Blotting.
Te preparation of cell lysate and western blotting were carried out as described previously [29] Te signifcance of diferences between two groups was evaluated with the unpaired t-test; diferences between multiple groups were evaluated with the one-way ANOVA and Tukey's post hoc test.Statistical signifcance was set at p < 0.05.

Cordycepin Induces Apoptotic Efects in OEC-M1 Cells through Activation of Caspases.
We have previously shown that cordycepin could induce apoptosis in OEC-M1 cells [29].Given that caspase cascades play critical roles in mediating apoptotic cell death [22], we frst examined whether caspases are involved in cordycepin-induced apoptosis in OEC-M1 cells.Tese results indicate that the activation of caspases is essential for cordycepin-induced apoptosis in OEC-M1 cells.

Cordycepin Induces Bid Cleavage in OEC-M1 Cells.
Te Bid is a proapoptotic protein in the Bcl-2 family, downstream of caspase-8.Caspase-8 activates Bid by cleaving it into t-Bid, which leads to the initiation of the mitochondrial pathway [22].After demonstrating that caspase-8 was activated by cordycepin, we next used western blotting to determine whether Bid is cleaved in cordycepintreated OEC-M1 cells (Figure 3(a)).Te quantitative analysis of the blots shows that cleavage of Bid signifcantly occurred at 24, 36, and 48 h treatments of cordycepin (Figure 3(b), p < 0.05).

Activation of JNK Is Required for Cordycepin-Induced
Apoptosis in Oral Cancer Cells.Te above experiments demonstrated that cordycepin-activated JNK, ERK, and p38 MAPK were present in OEC-M1 cells (Figure 4).Next, we used selective inhibitors of JNK (SP600125), ERK (PD184352), and p38 MAPK (SB202190), to determine which of these MAPKs was the major mediator of cordycepin-induced apoptosis in OEC-M1 cells.We found that, among the inhibitors, only the JNK inhibitor SP600125 could remarkably reduce the cordycepin-induced release of cytokeratin 18 fragments, a marker of apoptosis (Figure 5(a), p < 0.05).We also verifed the role of JNK activation in cordycepin-induced apoptosis in an independent way, by showing that treatment with SP600125 could signifcantly reduce cordycepin-induced DNA fragmentation, another indicator of apoptosis (Figure 5(b)).For an even stronger independent way to verify the role of JNK in cordycepininduced apoptosis, we treated OEC-M1 cells with JNK6o, a more selective inhibitor of JNK than SP600125.Treatment with JNK6o also abolished the release of cytokeratin 18 fragments induced by cordycepin (Figure 5(c), p < 0.05).Furthermore, we reproduced the same results using the same treatments with cordycepin and SP600125 or JNK6o in OC3 cells, another oral cancer cell line derived from buccal epidermal carcinoma.We found that treatment with SP600125 or JNK6o both signifcantly inhibited the cordycepin-induced release of cytokeratin 18 fragments from OC3 cells (Figure 5(d), p < 0.05).Together, these data represent strong evidence that activation of JNK plays an essential role in cordycepin-induced apoptosis in oral cancer cells.
We have shown that cordycepin-activated caspases (Figure 3), as well as JNK (Figure 4) and that JNK was required for cordycepin-induced apoptosis (Figure 5) in OEC-M1 cells.Given that JNK is involved in promoting caspase activation [26], we tested whether JNK is the upstream molecule of caspase activation induced by cordycepin by

Summary Chart of the Molecular Pathways Implicated with Cordycepin-Induced Apoptosis in OEC-M1 Cells.
To summarize the above results, we propose a hypothetical network of the signaling pathways as a model of the molecular mechanism underlying cordycepin-induced apoptosis in OEC-M1 cells (Figure 7).Te cascade of activation in the network begins with cordycepin-activated JNK, which contributes to caspase-8 and caspase-9 activations.Activation of caspase-8 may lead to Bid cleavage, contributing to activation of the mitochondrial pathway.Activation of both caspase-8 and caspase-9 results in activation of caspase-3, which promotes PARP cleavage and, ultimately, to apoptosis in OEC-M1 cells.

Discussion
We used controlled protein expression analysis to explore the molecular basis of cordycepin-induced apoptosis in oral cancer cell cultures.First, we showed that Z-VAD-FMK (a pan-caspase inhibitor) efectively inhibited cordycepin-  (-) or with 100 μM cordycepin (+) for 3, 6, 12, 24, 36, and 48 h, respectively.Protein expression levels were visualized using western blotting for phosphorylated JNK, total JNK, phosphorylated ERK, total ERK, phosphorylated p38 MAPK, total p38 MAPK, and β-actin (internal control).Protein expression levels were quantifed using the Quantity One image analysis system for (b) activated JNK, (c) activated ERK, and (d) activated p38 MAPK.* p < 0.05 vs. control.6 Evidence-Based Complementary and Alternative Medicine induced apoptosis (Figure 1), indicating that cordycepin induces apoptosis through activating caspases in OEC-M1 cells.Second, we showed that cordycepin treatment induced caspase-8 and caspase-9 activation in these cells (Figure 2), strongly suggesting that cordycepin may induce apoptosis through both the external and internal apoptosis pathways in OEC-M1 cells.Tird, we showed that cordycepin treatment leads to the activation (cleaving) of Bid (Figure 3), a proapoptotic Bcl-2 protein.Since the Bid is cleaved by caspase-8, contributing to mitochondrial pathway activation [22], we speculate that the death receptor may connect with the mitochondrial pathway to induce cell apoptosis under cordycepin treatment in OEC-M1 cells.Fourth, we showed that the activation of JNK was essential for cordycepininduced apoptosis in OEC-M1 cells.While cordycepin-activated many MAPKs, including JNK, ERK, and p38 MAPK (Figure 4), only the inhibition of JNK (by SP600125), but not the other MAPKs, could efectively inhibit cordycepin-induced apoptosis (Figure 5).Tis fnding is consistent with previous studies, which also showed that JNK plays a vital role in mediating apoptosis in lung cancer cells and ovarian cancer cells, respectively [30,31].Fifth, we showed that in cells where JNK was inhibited, the activation of caspase-8 and caspase-9 was also inhibited (Figure 6), placing JNK to the upstream in the cascade of caspase-8 and caspase-9 activation in OEC-M1 cells.However, it remains elusive how JNK activates the initiator caspase-8 and caspase-9 under cordycepin treatment in OEC-M1 cells.
It has been demonstrated that the death receptor pathway is activated when Fas Ligand (FasL), a death factor, binds its receptor, Fas [32].Since JNK reportedly promotes the expression of FasL [33], it is possible that JNK activates caspase-8 through the FasL/Fas death receptor pathway to induce cancer cell apoptosis.On the other hand, JNK could induce cancer cell apoptosis by activating proapoptotic Bcl-2 proteins Bax and Bim [34,35] that contribute to caspase-9 activation in the mitochondrial pathway [22,36].Tus, we speculate that cordycepin could stimulate JNK, which would then simultaneously activate both the death receptor pathway and the mitochondrial pathway to induce OEC-M1 cell apoptosis.
Tere is evidence that the ERK pathway, in addition to promoting apoptosis, might have a role associated with cell survival [37].In fact, we found that ERK inhibition signifcantly enhanced cordycepin-induced apoptosis in OEC-M1 cells, in sharp contrast to the efect of JNK inhibition (Figure 5(a)), consistent with the ERK function serving as a survival pathway during cordycepin treatment.In fact, studies have shown that treatment with ERK inhibitors could enhance tamoxifen-induced apoptosis and crizotinibinhibited tumor growth [38,39].Together, these lines of evidence suggest that blocking ERK signaling could be an excellent strategy for enhancing cordycepin-induced apoptosis in OEC-M1 cells, on the condition that targeted future in vitro and in vivo experiments will corroborate this scenario.
Te tumor suppressor p53 gene is important in the regulation of apoptosis [40], promoting the activation of Bid and caspases [41,42] or upregulating the expression of proapoptosis-related proteins such as Bax and PUMA [43,44].Previously, it was shown that JNK activated p53 to promote apoptosis [45].However, we showed that cordycepin promoted the activation of caspases (Figure 2) and Bid (Figure 3) to induce apoptosis in OEC-M1 cells, a p53 mutation cell line, discounting the scenario that cordycepininduced apoptosis depended on the p53 pathway.
Te molecular pathway through which cordycepin induces the activation of JNK in OEC-M1 cells remains to be elucidated.One study has reported that cordycepin promoted the production of reactive oxygen species (ROS) [46], which is a factor known to induce the activation of apoptosis signal-regulating kinase 1 (ASK1), an upstream molecule of   Evidence-Based Complementary and Alternative Medicine JNK [47].In an alternative scenario, cordycepin could activate AMP-activated protein kinase (AMPK) [48], which is also known to promote the activation of JNK [49].Tus, under a highly possible scenario, cordycepin activates JNK by activating the ROS-ASK1 and/or AMPK pathways to induce apoptosis in OEC-M1 cells, but further studies are necessary to work out the details of this mechanism.

Conclusion
Cordycepin activates JNK and the caspase pathways to induce apoptosis in OEC-M1 cells.Tese results can be used to design more successful therapy regimes combined with cordycepin anticancer efects to improve the prognosis of patients with oral cancer.

Figure 1 (
a) shows that treatment with Z-VAD-FMK, a pan-caspase inhibitor that can inhibit caspase activation, could block cordycepin-induced DNA fragmentation.It has been shown that caspase-cleaved cytokeratin 18, a biochemical marker of apoptosis, is released from epithelial cells during apoptosis[23].Te analysis by ELISA showed that cordycepin boosted the amount of cytokeratin 18 fragments released by OEC-M1 cells signifcantly above control levels (Figure1(b), p < 0.05), but this efect was fully reversed by treatment with the caspase inhibitor Z-VAD-FMK (Figure 1(b), p < 0.05).

Figure 1 :
Figure 1: Cordycepin induces apoptotic efects in OEC-M1 cells through the activation of caspases.OEC-M1 cells were treated without (control) or with 100 μM cordycepin and concurrently treated with the solvent control DMSO (D) or a 10 μM Z-VAD-FMK, an irreversible pan-caspase inhibitor (Z).(a) DNA fragmentation was assessed by using the DNA laddering assay.(b) Cytokeratin 18 fragment concentration in the cell culture supernatants was determined by ELISA.* p < 0.05 vs. control + DMSO group; # p < 0.05 vs. cordycepin + DMSO group.

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Evidence-Based Complementary and Alternative Medicine of signifcant expression of cleaved PARP at 36 and 48 h after cordycepin treatments of OEC-M1 cells (Figure 2(e), p < 0.05).